APX-115A, a pan-NADPH oxidase inhibitor, induces caspase-dependent cell death by suppressing NOX4-ROS signaling in EBV-infected retinal epithelial cells
Seung-Woo Hong, Min Hye Noh, Yeong Seok Kim, Dong-Hoon Jin, Sung Hwan Moon, Jae Wook Yang & Dae Young Hur
To cite this article: Seung-Woo Hong, Min Hye Noh, Yeong Seok Kim, Dong-Hoon Jin, Sung Hwan Moon, Jae Wook Yang & Dae Young Hur (2020): APX-115A, a pan-NADPH oxidase inhibitor, induces caspase-dependent cell death by suppressing NOX4-ROS signaling in EBV- infected retinal epithelial cells, Current Eye Research, DOI: 10.1080/02713683.2020.1718164
To link to this article: https://doi.org/10.1080/02713683.2020.1718164
Accepted author version posted online: 17 Jan 2020.
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Publisher: Taylor & Francis
Journal: Current Eye Research
DOI: 10.1080/02713683.2020.1718164
APX-115A, a pan-NADPH oxidase inhibitor, induces caspase-dependent cell death by suppressing NOX4-ROS signaling in EBV-infected retinal epithelial cells
Running Head : APX-115A induces apoptosis in EBV-retina cells
Seung-Woo Hong1,2, Min Hye Noh1, Yeong Seok Kim1, Dong-Hoon Jin2, Sung Hwan Moon3, Jae Wook Yang4, Dae Young Hur1*
1 Department of Anatomy, Inje University College of Medicine, 75 Bokji-ro, Busanjin- gu, Pusan 47392, Republic of Korea
2 Department of Convergence Medicine, University of Ulsan College of Medicine, Asan Medical Center, Olympic-ro 43-gil, Songpa-gu, Seoul, Republic of Korea
3 AptaBio Therapeutics Incorporation, Heungdeok 1-ro, Giheung-gu, Yongin-si, Gyeonggi-do, 16954, Republic of Korea
4 Department of Ophthalmology, Inje University Pusan Paik Hospital, 75 Bokji-ro, Busanjin-gu, Pusan 47392, Republic of Korea
Corresponding authors:
Dae Young Hur, Department of Anatomy, College of Medicine, Inje University, Bokji-ro 75, Busanjin-gu, Busan, Republic of Korea 614-735, Tel 82-51-890-6634, E-mail: [email protected]
Abstract
Purpose: Epstein-Barr virus is a -herpes virus that infects primary B cells and can transform infected cells into immortalized lymphoblastoid cell lines (LCL). The role of EBV in
malignancies such as Burkitt’s lymphoma and nasopharyngeal carcinoma is well understood, however, its role in EBV-infected retinal cells remains poorly understood. Therefore, we investigated the effect of EBV on the growth of retinal cells.
Methods: Previously, we established and reported a cell line model to address the relationship between EBV infection and retinal cell proliferation that used adult retinal pigment epithelium (ARPE-19) and EBV infection. To determine the effect of EBV on ARPE-19 cells, cell death was measured by propidium iodine/annexin V staining and reactive oxygen species (ROS) were measured by FACS, and protein expression was evaluated using western blot analysis. Also, downregulation of LMP1 and NADPH oxidase 4 (NOX4) expression was accomplished using siRNA technology.
Results: We found that ROS were dramatically increased in EBV-infected ARPE19 cells (APRE19/EBV) relative to the parental cell line. Additionally, the expression level of NOX4, a main source of ROS, was upregulated by EBV infection. Interestingly, downregulation of LMP1, one of the EBV viral onco-proteins, completely decreased EBV-induced ROS accumulation and the upregulation of NOX4. Treatment with APX-115A, a pan-NOX inhibitor, induced apoptotic cell death of only the EBV-infected ARPE19 cells but not the parental cell line. Pretreatment with z-VAD, a pan-caspase inhibitor, inhibited NOX inhibitor- induced cell death in ARPE19/EBV cells. Furthermore, APX-115A-induced cell death mediated the activation of JNK and ERK. Finally, we confirmed the expression level of NOX4, and APX-115A induced cell death of EBV-infected human primary retina epithelial cells and the activation of JNK and ERK.
Conclusion Taken together, these our results suggest that APX-115A could be a therapeutic agent for treating EBV-infected retinal cells or diseases by inhibiting LMP1-NOX4-ROS signaling.
Key words: Retinal pigment epithelial cell, EBV-infection, LMP1, NOX4, NOX inhibitor
Introduction
Epstein-Barr virus was initially discovered in Burkitt’s lymphoma patients and was classified as a -herpes onco-virus [1]. EBV infection is ubiquitous, infecting approximately 90% of the world adult population [2]. The oncogenic effect of EBV has been well studied in various malignancies, such as Burkitt’s lymphoma, non-Hodgkin’s lymphoma, nasopharyngeal carcinoma, and gastric cancer [3-6]. Although cases of EBV infection are rarely detected in retinal diseases, EBV is associated with ocular malfunction, especially chronic uveitis [7-9]. However, the detailed molecular mechanism underlying the effect of EBV in retinal diseases or in retinal cell models remains elusive. Understanding these mechanisms, which could provide a novel potential target for EBV-induced chronic ocular diseases, may result in new therapies for ocular diseases.
Reactive oxygen species (ROS) are involved in various cell homeostasis functions, including cell proliferation, apoptosis, invasion, angiogenesis, and the progress of virus- induced malignant transformation [10-12]. Recently, it was reported that increases in ROS level by EBV infection provide a growth advantage to EBV-infected cells by inducing expression of cell proliferation-associated genes [13]. Moreover, with respect to the role of ROS in retinal diseases, such as diabetic retinopathy, it has been reported that neovascularization is accelerated through the secretion of VEGF (vascular endothelial growth factor) by ROS [14]. Therefore, ROS is a potential therapeutic target in virus-infected cancers or retinal diseases. It has reported that LMP1 regulates the expression NOX and NOX regulatory subunit p22phox [15].
The NADPH oxidase (NOX) family, is composed of the NOX1, 2, 3, 4, 5 and dual oxidase (DUOX) 1, 2 proteins and includes the major enzymes responsible for the production
of reactive oxygen species [16, 17]. Recently, overexpression of NOX isoform enzymes has been reported in non-small cell lung cancer and pancreatic cancer [18, 19]. Furthermore, these enzymes play an important role in retinal diseases associated with diabetic retinopathy and diabetic nephropathy through increases in ROS, resulting in the induction of neovascularization [20, 21].
ROS-induced cancer cell death is deeply associated with MAPK pathway. ROS produced by both extrinsic and intrinsic stimuli, is involved in MAPK activation through various mediators including protein kinase C [22]. Subsets of MAPK pathway are ERK(extracelluar signal regulated kinases), JNK(c-jun N-terminal kinase), and p38 MAPK were, which were activated by phosphorylation by MAPK kinases. These MAPK pathway provoke transcriptions of several factors linked to various cell responses such as cell growth, inflammation, apoptosis and cell death [23].
Previously, we reported that cell morphological changes associated with the epithelial to mesenchymal transition were induced in EBV-infected ARPE-19 cells, a retinal in vitro cell model system [24]. In this study, we showed that the ROS level in EBV-infected ARPE-19 cells was dramatically enhanced by the LMP1-NOX4 signaling pathway. Moreover, inhibition of ROS by treatment with APX-115A, a pan-NOX inhibitor, in EBV-infected ARPE-19 cells induced caspase-dependent apoptosis by activating the ERK-JNK pathway.
Materials and methods
Cell cultures and reagents
Human adult retinal epithelial 19 (ARPE-19) cells were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). Primary human retina pigment epithelial (HRPEpi) cells were purchased from ScienCell (Carlsbad, CA, USA) [24]. Previously, we
established for EBV-infected APRE-19 cells [24]. The virus titer of B95-8 EBV virions from B95-8 cell line (ATCC) was >102 transforming units/mL. The transforming units were determined by lumping formation of peripheral mononuclear cells after infection using infectious culture supernatant. After ARPE-19 cells (2 x 105 cells/T25 flask/4 mL media) were completely attached, an equal volume of EBV supernatant (4 mL, 75 ± 11 colony- forming units per milliliter) was added. These cultures were maintained to visualize the expression of EBV latent genes for 4 weeks. Parental ARPE-19 cells or EBV-infected ARPE-
19 cells were cultured in DMEM/F12 medium (Mediatech Inc., Corning Subsidiary, Manassas, VA, USA) or RPMI-1640 medium (Corning) containing 10% heat-inactivated fetal bovine serum (Tissue Culture Biologicals, Tulare, CA, USA), 100 IU/ml penicillin, and 100 µg/ml streptomycin at 37°C in a 5% CO2 humidified atmosphere. Z-VAD-FMK (Sigma, St. Louis, USA), SP600125 (Selleck Chemicals, Houston, TX, USA) and PD98059 (Selleck Chemicals) were used for inhibitor treatment. APX-115A, a pan-NADPH oxidase inhibitor, was kindly provided and synthesized by AptaBio Therapeutics Incorporation (Yongin, Republic of Korea).
siRNA transfection, drug treatments and cell death
Cells were seeded at 5X105 cells in 60mm3 dishes, and then transfected with scramble siRNA (5’-GCU UUG GGA UAU CAU AGC GAU GAA U-3’) or NOX4 siRNA (5’-GUC AAC AUC CAG CUG UAC C-3’) or LMP1 siRNA (5’-AAG AGC CUU CUC UGU CCA CU-3’)
for 48 h using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according’s to recommended guide lines. All siRNAs were provided by Genolution Pharmaceutical Inc. (Seoul, Republic of Korea). Cells were treated with APX-115A, a pan-NADPH oxidase
inhibitor, at the indicated doses for 48 h. The rate of cell death was analyzed using trypan blue exclusion method.
ROS staining
Cells were transfected with NOX4 siRNA or LMP1 siRNA for 48 h or treated with APX- 115A at the indicated doses for 48 h. To detect ROS levels of transfected or APX-115A treated cells, cells were incubated with 20 M H2DCF-DA (Sigma) for 30 min, washed with PBS, trypsinized and then harvested by 500 l PBS. DCF-stained cells were measured by fluorescence-activated cell sorting (FACS; BD Biosciences) using Cell Quest 3.2 (Becton Dickson) software for analysis.
Western blot analysis
Cells were lysed with RIPA lysis buffer (50 mM HEPES pH7.4, 150 mM NaCl, 1 mM EDTA,
2.5 mM EGTA, 1 mM DTT, 0.1 % Triton X-100) containing a protease and phosphatase inhibitor (Sigma). After lysis process, we sonicate for 10 seconds to shear DNA. And then, cell lysates (20 µg per lane) were resolved by 8~15% SDS-PAGE (sodium dodecyl sulfate- polyacrylamide gel electrophoresis) and then transferred to an Immobilion PVDF membrane (EMD Millipore, Billerica, MA, USA). The membrane was blocked with 5% nonfat skim milk in TBS-T buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1 % Tween 20) and incubated with following primary antibodies at 4C overnight: anti--actin, anti-EBNA1, anti-EBNA3C, anti-NOX2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-EBNA2 (Abcam, Cambridge, MA, USA), anti-LMP1 (Abnova, Taipei, Taiwan), anti-cleaved caspase- 9, anti-cleaved caspase-3, anti-phospho-JNK, anti-JNK, anti-phospho-ERK, anti-ERK (Cell
Signaling Technology, Beverly, MA, USA), anti-NOX4 antibody was kindly provided from Dr. Bae (Department of Life Science, Ewha Womans University, Republic of Korea). Bound primary antibodies were detected using horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Cell Signaling) and chemiluminescence detection reagents (Amersham, Buckinghamshire, UK).
Statistical analysis
All datas are shown as the mean SD. A two-sided t-test was performed to evaluate the P value, and P<0.05 was considered statistically significant.
Results
EBV-infected ARPE-19 cells dramatically accumulates ROS by upregulating the expression of NOX4
EBV-infected ARPE-19 cells were shown to grow more quickly than parental ARPE-19 cells. To further investigate for theses difference, we focused on the association between EBV- infection and ROS. To achieve this aim, we initially compared the levels of ROS in the parental ARPE-19 cells and in EBV-infected ARPE-19 cells. Interestingly, the level of ROS determined by FACS analysis was significantly increased by EBV infection (Fig. 1a). To investigate this increase of ROS content upon EBV infection, we analyzed the expression levels of the ROS-generating enzyme NOX family. Surprisingly, expression of the NOX4 protein was dramatically increased in EBV-infected ARPE-19 cells but not in parental ARPE- 19 cells (Fig. 1b). Next, we examined the effect of NOX4 expression on the viability of EBV- infected ARPE-19 cells using flowcytometric analysis. Downregulation of NOX4 expression using siRNA technology dramatically increased annexin-V+/PI- cells, annexin-V+/PI+ cells,
and annexin-V-/PI+ cells in EBV-infected ARPE-19 cells in a dose-dependent manner, whereas no increase was noted in ARPE-19 parental cells (Fig. 1c). Moreover, NOX4 siRNA treatment induced the cleavage of caspase-3 in a dose-dependent manner (Fig. 1d). In addition, knockdown of NOX4 expression decreased the level of ROS in EBV-infected ARPE-19 cells (Fig. 1e). These results demonstrate that the inhibition of the increase ROS due to NOX4 upregulation by EBV infection induces cell death in ARPE-19 cells.
The block of ROS signaling through the regulation of NOX4 expression by LMP1 induces cell death in EBV-infected ARPE-19 cells
As shown Fig.1, NOX4 expression was increased by EBV infection. Based on this data, we analyzed the effect of LMP1 expression on NOX4 level. The knockdown of LMP1 expression by siRNA transfection was confirmed by western blot analysis. The level of NOX4 following LMP1 downregulation was significantly decreased in a siRNA concentration-dependent manner (Fig. 2a). Consistent with this finding, the population of dead cells increased in EBV-infected ARPE-19 cells transfected with LMP1 siRNA (Fig. 2b). In addition, inhibition of LMP1 expression decreased the level of ROS in EBV-infected ARPE-19 cells (Fig. 2c). Our results indicate that LMP1 modulates the expression of NOX4, which regulates the ROS level to promote cell viability in EBV-infected ARPE-19 cells.
APX-115A induces caspase-dependent apoptosis in only EBV-infected ARPE-19 cells Blocking ROS in EBV-infected ARPE-19 cells induced cell death (Figs. 1 and 2). Therefore, a pharmacological NOX inhibitor would only induce cell death in EBV-infected cells. Recently, our co-workers identified APX-115A as a pan-inhibitor of NOX enzymes [23]. To
test this hypothesis, we first examined the effects of APX-115A in ARPE-19 cells and EBV- infected ARPE-19 cells. Treatment with APX-115A dose-dependently increased apoptotic cell death in EBV-infected ARPE-19 cells, whereas cell death was not increased in ARPE-19 parental cells (Fig. 3a). This result implies that APX-115A affects cellular ROS levels only in EBV-infected cells. To confirm this hypothesis, we analyzed the content of ROS after treatment with APX-115A. ROS levels in EBV-infected APRE-19 cells dose-dependently decreased after treatment with APX-115A (Fig. 3b). Next, we determined whether cell death in response to APX-115A was caspase-dependent. For this experiment, we analyzed the cleavage active forms of caspases in APX-115A-treated ARPE-19 cells by western blot. The cleavage active forms of caspase-3 and -9 was clearly detected in response to APX-115A in a dose-(Fig. 3c) and time-dependent manner (Fig. 3d). Consistent with this data, treatment of EBV-infected ARPE-19 cells with z-VAD, a pan-caspase inhibitor, completely inhibited APX-115A-induced cell death (Figs. 3e and f). These results indicate that APX-115A induces caspase-dependent cell death by inhibiting the NOX4-ROS signaling pathway.
APX-115A activates cell death by the phosphorylation of ERK and JNK in EBV-infected ARPE-19 cells
Based on these reports, we examined the effects of MAPK molecules on APX-115A-induced cell death. Among these MAPK pathway molecules, phosphorylation of JNK and ERK gradually was increased after exposure to APX-115A in a dose dependent manner in EBV- infected APRE-19 cells (Fig. 4a). To further confirm this result, we analyzed the effect of JNK or ERK inhibitor on APX-115A-induced cell death. Cell death analysis demonstrated that inhibition of JNK or ERK completely suppressed APX-115A-induced cell death in EBV-
infected ARPE-19 cells (Fig. 4b). These results suggest that activation of JNK and ERK is
indispensable for APX-115A-induced cell death. We additionally evaluated the relationship of JNK and ERK with APX-115A-induced cell death. To perform these experiments, we analyzed the phosphorylation level of JNK in ERK inhibitor-treated cells and the activation of ERK in response to a JNK inhibitor in EBV-infected APRE-19 cells, respectively. Phosphorylation of JNK was inhibited in both ERK and JNK inhibitor-treated cells, whereas activation of ERK was suppressed only in ERK inhibitor-treated cells (Figs. 4c and 4d). This result indicates that JNK is sequentially phosphorylated by ERK phosphorylation after APX- 115A treatment.
APX-115A also induces cell death and the phosphorylation of ERK and JNK in EBV- infected human primary retina epithelial cells (HRPEpi) cells
We also developed EBV-infected HRPEpi cells to determine whether human primary retina epithelial cells show similarly transformation and the expression of NOX4. As expected, the expression of the NOX4 protein was also increased in EBV-infected HRPEpi cells compare to parental HRPEpi cells (Fig. 5a). And treatment with APX-115A dose-dependently increased apoptotic cell death in EBV-infected HRPEpi cells, whereas cell death was not increased in parental HRPEpi cells (Fig. 5b). And, the cleavage active forms of caspase-3 and
-9 was clearly detected in response to APX-115A in a dose-dependent manner (Fig. 5c). Moreover, the phosphorylation of JNK and ERK was also increased after exposure to APX- 115A in a dose dependent manner in EBV-infected HRPEpi cells (Fig. 5d).
Discussion
Several studies have reported that infection by tumor viruses is associated with increased oxidative stress, which exerts an important role in virus-induced transformation [26, 27]. Thus, an inhibitor of ROS may be an effective agent for virus-associated diseases. Recently, it was reported that EBV increases the proliferation of EBV-infected cells through the induction of ROS by cellular growth-associated factors in B lymphocytes [13]. Although EBV infection are rare in retinal diseases, the effect of the EBV to RPE cells when it is active has been identified clinically as uveitis, retinitis, retinopathy, and acute retinal necrosis [7-9]. However, the molecular mechanisms underlying cellular responses to EBV infection are poorly understood in retinal-diseases or in a retinal cell line model, although the ratio of its infection is very low. Here, using the retinal pigment epithelial cell line ARPE-19 and human primary retina epithelial (HRPEpi) cells, we demonstrated for the first time that the increase in ROS mediated by the EBV-encoded LMP1 protein and the cellular ROS-generating enzyme NOX4 pathway play pivotal roles in the maintenance for EBV-induced retinal cell viability. In addition, we showed that APX-115A, a pan-NADPH oxidase inhibitor, induces cell death (both apoptosis and necrosis were involved in the cell death) in EBV-infected retinal cells, but not in EBV-negative parental cells, by activating the ERK- and JNK-caspase dependent apoptotic signaling axis.
To date, searches have been performed to identify target molecules for the treatment of virus-infected retinal diseases or virus-infected cancers. However, the identification of the viability maintenance factors associated with the virus that supports the cellular viability link in EBV-infected retinal diseases or in a cell line model remained elusive. It was reported that EBV-infected B lymphocytes enhance cell proliferation by upregulating ROS [28, 29]. In this study, we focused on the relationship between ROS-generating enzymes and EBV infection.
First, we investigated whether EBV infection could regulate the level of ROS and the expression of the ROS-generating enzyme NOX family using an EBV-infected retinal cell line model system. Based on our results, we focused on the effect of NOX4 expression on the viability of EBV-infected cells. Inhibition of NOX4 in EBV-infected cells led to cell death in accordance with the decrease in ROS content. Therefore, ROS modulation via regulation of NOX4 expression by EBV infection could be a target for treatment in EBV-infected retinal cells. We further examined the relationship between the EBV latency genes and NOX4 expression in EBV-infected retinal cells. Recently, it was reported that the EBV oncoprotein LMP1 regulates the expression NOX and a NOX regulatory subunit [15]. As shown in Fig. 2A, LMP1 regulated the expression of NOX4, suggesting that, in EBV-infected cells, ROS is regulated by the LMP1-NOX4 pathway. We also analyzed the effect of LMP1 downregulation on EBV-infected cells. The percentage of dead cells dramatically increased in LMP1-inhibited cells, whereas the levels of ROS decreased.
NOX enzyme inhibitors represent a new potential anti-cancer agent in various cancers, such as non-small cell lung cancer and pancreatic cancer [18, 19]. However, whether there is a differential cellular response to NOX inhibitors in virus-infected versus un-infected cells has not been determined, especially in retinal cell models. Interestingly, NOX enzymes have been reported to enhance neovascularization by upregulating ROS levels in retinal blindness associated with diabetic retinopathy or diabetic nephropathy [19, 20]. Recently, our co- workers developed the NOX inhibitor APX-115A as an intravitreal shape. In this study, we showed that APX-115A, a pan-NOX inhibitor, induced cell death in EBV-infected retinal cells, but not parental cells, through the modulation of ROS. To further study the mechanism of APX-115A-induced cell death, we examined the cleavage active forms of caspases after
exposure to APX-115A. Treatment of cells with APX-115A resulted in the cleavages of caspase-9 and caspase-3 in EBV-infected RPE cells, indicating that APX-115A induces caspase-dependent mitochondria-mediated apoptosis.
One major pathway of ROS-induced cell death is MAPK signaling, which plays a prominent role in cell homeostasis [30]. Inhibition of JNK, ERK, or p38 under conditions of various apoptotic stimuli inhibits ROS-induced cell death by modulating MAPK-downstream molecules. Thus, we addressed whether MAPK affects cell death induced by APX-115A in EBV-infected retinal cells. Phosphorylation of JNK and ERK was induced upon to exposure to APX-115A, suggesting that MAPK is associated with the cell death induced by APX-115A. We also confirmed that inhibition of JNK and ERK suppresses apoptotic cell death induced by APX-115A. In addition, inhibition of ERK led to decreased JNK phosphorylation in addition to decreased phosphorylation of ERK. These results indicate that ERK activation works upstream of JNK during APX-115A induced apoptotic cell death.
The recent development of drug for treating diseases and cancers associated with virus infection has established a new anti-viral technology. In this study, we clearly demonstrated the role of ROS through LMP1-NOX4 signaling in EBV-infected retinal cells, and that APX- 115A could be a potential drug for treating EBV-infected retinal cells. However, the detailed regulatory mechanisms of LMP1 and NOX4 were not addressed, and massive RPE cell death could lead to loss of retinal function, and lead to retinal degeneration. Therefore, we should be cautious regarding the implication of the inhibitor in the eye. Our results suggest that APX-115A is a potent therapeutic agent for EBV-associated retinal diseases including cancer that functions through the modulation of the NOX enzyme.
Declaration of Interest
Sung Hwan Moon works at Aptabio Therapeutics Inc, Republic of Korea, which develops APX-115A. The remaining authors declare no conflict of interest.
Acknowledgments
This study was supported by a grant from the Korea Healthcare Technology R&D Project, Ministry of Health and Welfare Affairs, Republic of Korea (grant no. HI15C2800).
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Figure legends
Fig. 1. NOX4-induced ROS accumulation caused by EBV affects ARPE-19 cell viability. (a) ARPE-19 and EBV-infected ARPE-19 cells were incubated with 20 M H2DCF-DA for 30 min, washed with PBS, trypsinized, and harvested in PBS for ROS analysis using FACS. (b) ARPE-19 and ARPE-19/EBV cells were subjected to western blot analysis for EBV- associated proteins and NOX family members. -actin was used as a loading control. EBV- infected ARPE-19 cells were transfected with NOX4 siRNA at the indicated concentrations for 48 h. (c) The cells were washed, and then stained with PI/Annexin-V. The graphs represent the mean ± S.E. of three independent experiments in duplicate. *P<0.05. (d) Levels of ROS were analyzed using FACS.
Fig. 2 Up-regulation of NOX4 expression by the EBV oncoprotein LMP1 results in the
accumulation of ROS. EBV-infected ARPE-19 cells were transfected with LMP1 siRNA for 48 h at the indicated doses. (a) Expression levels of LMP1 and NOX4 were evaluated by western blot analysis. -actin was used as a loading control. (b) The ratio of cell death was determined using PI/Annexin-V staining method in LMP1 siRNA-transfected cells. The data represent the mean ± S.E. of three independent experiments in duplicate. *P<0.05. (c) LMP1 siRNA-transfected cells were incubated with 20 M H2DCF-DA for 30 min, washed with PBS, trypsinized, and collected in PBS for FACS analysis. The results are representative of three independent experiments.
Fig. 3 APX-115A, a pan-NOX inhibitor, induces caspase-dependent cell death by blocking ROS only in EBV-infected ARPE-19 cells. (a) ARPE-19 and ARPE-19/EBV cells were treated with APX-115A at the indicated doses for 48 h, and then cell death was measured using PI/Annexin-V staining. Each value represents the mean ± S.E. of three independent experiments in duplicate. **P<0.01. (b) EBV-infected ARPE-19 cells were treated with 5, 10, and 20 M APX-115A for 48 h, and cells then were stained with 20 M H2DCF-DA for 30 min, followed by ROS analysis by FACS. (c) EBV-infected ARPE-19 cells were treated with APX-115A at the indicated doses for 48 h. Cell lysates were used for western blot analysis using anti-cleaved caspase-3 or anti-cleaved caspase-9 antibodies. (d) Cells were treated with 20 M APX-115A at the indicated times, and western blot analysis was then performed. (e) Cells were pretreated with 50 M z-VAD-fmk, a pan-caspase inhibitor, before treatment with 20 M APX-115A. Cell death was analyzed using PI/Annexin-V staining method. The data represents the mean ± S.E. of three independent experiments in duplicate. **P<0.01. (f)
Activation of caspase-3 and caspase-9 was confirmed by western blot analysis using anti-
cleaved caspase-3 or anti-cleaved caspase9 antibodies. The results are representative of three independent experiments.
Fig. 4 Cell death caused by APX-115A activates the expression of JNK and ERK in EBV- infected ARPE-19 cells. (a) EBV-infected ARPE-19 cells were treated with APX-115A at the indicated doses for 48 h. Activation of JNK and ERK was evaluated by western blot analysis using anti-p-JNK or anti-p-ERK antibodies. (b) Cells were pretreated with 20 M SP600125 or 20 M PD98059 1 h before treatment with 20 M APX-115A. After 48h, cell death was measured using PI/Annexin-V staining method. The values represent the mean ± S.E. of three independent experiments. **P<0.01. (c, d) Cells were treated as in Fig. 4b, and the JNK, ERK, and caspase-3 protein levels were determined by western blot. The results are representative of three independent experiments.
Fig. 5 EBV infection induces NOX4 expression, the treatment of APX-115A induces caspase- dependent cell death and the expression of JNK and ERK in EBV-infected HRPEpi cells. (a) HRPEpi and HRPEpi/EBV cells were subjected to western blot analysis for retina protein (RPE65), EBV-associated proteins, and NOX family members. -actin was used as a loading control. (b) HRPEpi and HRPEpi/EBV cells were collected and washed, and then stained with trypan blue. And the ratios of cell death were analyzed. The graphs represent the mean ±
S.E. of three independent experiments in duplicate. **P<0.01. (c) EBV-infected HRPEpi cells were treated with APX-115A at the indicated doses for 48 h. Cell lysates were used for western blot analysis using anti-cleaved caspase-3 or anti-cleaved caspase-9 antibodies. P.C.;
positive control. (d) Cells were treated as in Fig. 5c. Activation of JNK and ERK was evaluated by western blot analysis using anti-p-JNK or anti-p-ERK antibodies. The HRPEpi is a human primary retina epithelial cell; RPE65 is an RPE-specific marker. The results are representative of three independent experiments.
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