Role of Peroxiredoxin 1 Induced by Epstein-Barr Virus Infection in Nasopharyngeal Carcinoma.

BACKGROUND/AIM: Nasopharyngeal carcinoma (NPC), a common cancer in Southern China, is associated with Epstein-Barr Virus (EBV) infection. Although many therapies for NPC have been established, the definite role of EBV in NPC remains unclear. Therefore, this work focuses on LMP2A, a latent EBV gene, and investigates whether LMP2A is related to peroxiredoxin 1 (PRDX1) in EBV-positive NPC. MATERIALS AND METHODS: The mRNA and protein expression levels of LMP2A, PRDX1, and beta-catenin were compared in patient samples. To identify molecular mechanisms, EBV-negative NP69 and EBV-positive C666-1 NPC cell lines were used. After making an agar cell block for cell slides, the intensity of LMP2A expression was observed visually. To measure the level of reactive oxygen species, both fluorescence microscope and flow cytometry were used. To investigate the intracellular signaling molecular mechanisms with and without the LMP2A gene, reverse transcription polymerase chain reaction and western blotting were used. RESULTS: Both patient samples and cells of nasopharyngeal carcinoma infected with EBV had increased expression of LMP2A compared with controls, and high ROS levels were identified. Cell viability assay showed that LMP2A promoted cell growth by regulating gene expression. Furthermore, LMP2A induced the expression of PRDX1 and beta-catenin. LMP2A also increased the expression of both cyclin B1 and cyclin D1. CONCLUSION: In NPC cells, PRDX1 and beta-catenin were regulated through LMP2A expression, which reduced cell growth through cell cycle-related gene expression. This study suggests that LMP2A could be a target molecule for inhibiting cancer progression in NPC cells infected with EBV.

Targeting IGF2-IGF1R Signaling to Reprogram the Tumor Microenvironment for Enhanced Viro-Immunotherapy.

BACKGROUND: The FDA approval of oncolytic herpes simplex-1 virus (oHSV) therapy underscores its therapeutic promise and safety as a cancer immunotherapy. Despite this promise, the current efficacy of oHSV is significantly limited to a small subset of patients largely due to the resistance in tumor and tumor microenvironment (TME). METHODS: RNA sequencing (RNA-Seq) was used to identify molecular targets of oHSV resistance. Intracranial human and murine glioma or breast cancer brain metastasis (BCBM) tumor-bearing mouse models were employed to elucidate the mechanism underlying oHSV therapy-induced resistance. RESULTS: Transcriptome analysis identified IGF2 as one of the top secreted proteins following oHSV treatment. Moreover, IGF2 expression was significantly upregulated in 10 out of 14 recurrent GBM patients after treatment with oHSV, rQNestin34.5v.2 (71.4%) (p=0.0020) (ClinicalTrials.gov, NCT03152318). Depletion of IGF2 substantially enhanced oHSV-mediated tumor cell killing in vitro and improved survival of mice bearing BCBM tumors in vivo. To mitigate the oHSV-induced IGF2 in the TME, we constructed a novel oHSV, oHSV-D11mt, secreting a modified IGF2R domain 11 (IGF2RD11mt) that acts as IGF2 decoy receptor. Selective blocking of IGF2 by IGF2RD11mt significantly increased cytotoxicity, reduced oHSV-induced neutrophils/PMN-MDSCs infiltration, and reduced secretion of immune suppressive/proangiogenic cytokines, while increased CD8+cytotoxic T lymphocytes (CTLs) infiltration, leading to enhanced survival in GBM or BCBM tumor-bearing mice. CONCLUSION: This is the first study reporting that oHSV-induced secreted IGF2 exerts a critical role in resistance to oHSV therapy, which can be overcome by oHSV-D11mt as a promising therapeutic advance for enhanced viro-immunotherapy.

APX­115A, a pan­NADPH oxidase inhibitor, reduces the degree and incidence rate of dry eye in the STZ­induced diabetic rat model.

Dye eye disease (DED) is a common ocular disorder in patients with diabetes. It has been reported that APX-115A, a pan-nicotinamide adenine dinucleotide phosphate (NAPDH) oxidase inhibitor, has an apoptosis-inducing effect on Epstein-Barr virus-infected retinal epithelial cells, but its effects in DED are poorly understood. Therefore, a rat model of diabetes was used in the present study to investigate whether APX-115A has an impact on DED in diabetic rats. A diabetic model was established in male Sprague Dawley rats via the intraperitoneal injection of streptozotocin. The eyeballs of the rats were treated with a solution containing APX-115A or a saline control. Tear secretion was measured with the phenol red thread tear test, and the morphology of the eyeball and lacrimal gland tissues was determined using hematoxylin and eosin staining. In addition, localization of NAPDH oxidase 2 (NOX2) in the eyeball and lacrimal gland tissues was detected by immunohistochemistry. The APX-115A treatment had no effect on body weight, blood glucose level or the size of the lacrimal glands. However, morphological changes, namely intracellular vacuoles and acinar atrophy, were observed in the lacrimal glands of the diabetic rats, and APX-115A treatment attenuated these changes. Immunohistochemistry revealed that NOX2 expression was decreased in the lacrimal glands of the diabetic rats, and APX-115A treatment did not attenuate the reduction in NOX2. The corneas of the diabetic rats treated with APX-115A exhibited no change in thickness but had lower NOX2 expression levels compared with those of the control diabetic rats. APX-115A also increased tear secretion and ameliorated the histological changes associated with diabetes. Furthermore, the NOX2 expression levels in the corneas of the diabetic rats treated with APX-115A were restored to the levels observed in normal rats. These findings suggest that APX-115A has potential as a therapeutic agent for DED.

Olaparib-induced Apoptosis Through EBNA1-ATR-p38 MAPK Signaling Pathway in Epstein-Barr Virus-positive Gastric Cancer Cells.

BACKGROUND: Epstein-Barr virus (EBV)-associated gastric cancer has been identified as a cancer subtype with definitive clinical and molecular characteristics. Although olaparib, a poly ADP ribose polymerase (PARP) inhibitor, is considered a potential effective agent for gastric cancer, the effect and underlying mechanism of olaparib on gastric cancer depending on EBV infection is not fully understood. MATERIALS AND METHODS: EBV-positive SNU719 and EBV-negative SNU638 gastric cancer cell lines were used to identify the effects of olaparib using the trypan blue exclusion method and annexin V staining assay. To observe the underlying cellular signaling mechanisms of olaparib-induced cell death, Epstein-Barr virus nuclear antigen 1 (EBNA1) and signaling related molecule expression were assessed using transfection, silencing of specific genes using small interfering RNA (siRNA), western blotting and signaling inhibition assay. RESULTS: Olaparib decreased the cell viability of EBV-positive SNU719 gastric cancer cells through caspase-3-dependent apoptosis in a dose dependent manner, whereas EBV-negative SNU638 gastric cancer cells showed drug resistance to olaparib. EBNA1 was expressed in SUN719 gastric cancer cells; however, ataxia telangiectasia and Rad3 related (ATR) and phosphorylated ATR kinase were expressed in SNU638 gastric cancer cells. EBNA1 transfection decreased ATR phosphorylation through p38 mitogen-activated protein kinase (MAPK) phosphorylation in SUN638 gastric cancer cells, and silencing of ATR kinase increased the susceptibility of these cells to olaparib treatment. Moreover, VE-821, an ATR kinase specific inhibitor, also increased the sensitivity of SNU638 cells to olaparib. In contrast, SB203580, a p38 MAPK inhibitor, inhibited this increase in sensitivity to olaparib by EBNA1 transfection. CONCLUSION: Olaparib treatment led to different cellular responses depending on EBV infection in gastric cancer cell lines. These results provide new insights into the mechanism of olaparib-induced apoptosis in gastric cancer cells and suggest that EBV infection should be considered when developing new potential therapeutic agents for gastric cancer.

Effects of Epstein-Barr Virus Infection on the Response of Human Breast Cancer Cells to Nicotine.

BACKGROUND/AIM: The purpose of our study was to test whether EBV infection affects the response of human breast cancer cells to nicotine. In addition, the underlying signaling mechanisms were evaluated. MATERIALS AND METHODS: EBV-infected MDA-MB-231 and LMP1-transfected MDA-MB-231 breast cancer cells were established. Reverse transcription-polymerase chain reaction and western blotting were performed to evaluate nicotine receptor expression. To verify the functional role and underlying signaling mechanism of nicotine receptor expression induced by EBV infection, morphologic analysis, and proliferation and inhibition assays were performed. RESULTS: Both EBV infection and LMP1 transfection increased cell proliferation and induced the up-regulation of α9-nAChR expression. Additionally, nicotine treatment induced tumorigenic activity in both EBV-infected and LMP1-transfected MDA-MB-231 breast cancer cells. Western blot and inhibitor assays showed that the nicotine-induced signaling was mediated through MAPK/ERK and AKT signaling pathways in EBV-infected and LMP1-transfected breast cancer cells, respectively. CONCLUSION: These results suggest that EBV infection and EBV-related LMP1 may act as potential molecular targets for breast cancer risk associated with nicotine, and provide a novel insight into the mechanism of nicotine stimulation in EBV-positive breast cancer cells.

Erlotinib Activates Different Cell Death Pathways in EGFR-mutant Lung Cancer Cells Grown in 3D Versus 2D Culture Systems.

BACKGROUND/AIM: Non-small cell lung cancer patients with epidermal growth factor receptor (EGFR) mutation have been shown to have a good response to erlotinib, a receptor tyrosine kinase inhibitor of EGFR. In this study, we found that the cell death pathways activated by erlotinib in 2D and 3D culture systems are different. MATERIALS AND METHODS: The cell death pathways induced by erlotinib were evaluated by flow cytometry and immunoblotting in both 2D and 3D culture systems of EGFR mutant lung cancer cells. RESULTS: Treatment with erlotinib induced caspase 8 activation and up-regulation of TNF-related apoptosis-inducing ligand (TRAIL) expression only in 3D cultures. Knockdown of TRAIL attenuated both erlotinib-induced activation of caspase-8 and apoptosis in 3D cultures. Erlotinib also increased LC3, an autophagy marker, expression and c-Jun N terminal kinase (JNK) activation. Both 3-MA as an autophagy inhibitor and SP600125 as a JNK inhibitor, significantly inhibited erlotinib-induced cell death. CONCLUSION: Erlotinib induces apoptotic cell death in 3D cultures through an autophagy-TRAIL-JNK pathway.

Celecoxib upregulates ULBP-1 expression in lung cancer cells via the JNK/PI3K signaling pathway and increases susceptibility to natural killer cell cytotoxicity.

Lung cancer has the highest cancer mortality rate in the world, and effective therapies are still required. Cyclooxygenase-2 (COX-2) is highly expressed in numerous types of cancer, and is therefore considered a possible target of cancer treatment. Celecoxib, a selective COX-2 inhibitor, has binding pockets that interact with COX-2 and disrupt its enzymatic activities. In addition, celecoxib is able to affect cellular functions in a COX-2-independent manner. The present study aimed to investigate if celecoxib affected natural killer (NK) cell receptors and susceptibility to NK cell toxicity. For this purpose, PCR, immunoblotting, flow cytometry analysis and NK cell cytotoxicity assays were performed. The present study revealed that sublethal concentrations of celecoxib increased the expression levels of UL16-binding protein 1 (ULBP-1), a natural-killer group 2 member D (NKG2D) ligand, in lung cancer A549 and H460 cell lines. ULBP-1 mRNA and protein expression was induced in a dose- and time-dependent manner after celecoxib treatment. Expression levels of other NKG2D ligands, such as ULBP-2, ULBP-3, MHC class I-related chain A (MICA) and MICB did not change considerably compared to ULBP-1 in response to celecoxib treatment. Fluorescence microscopic images revealed abundant ULBP-1 in the cytoplasm after celecoxib treatment. Both JNK and PI3K may be involved in the induction of ULBP-1 expression after celecoxib treatment in A549 and H460 cells. In a NK cytotoxicity assay, celecoxib increased the sensitivity to NK cell-mediated cytotoxicity via interaction with ULBP-1 in lung cancer cells. Overall, the present results demonstrated that celecoxib treatment induced ULBP-1 expression in lung cancer cells, thereby increasing their susceptibility to NK cell cytotoxicity. These results suggest that the effects of conventional anticancer therapy may potentially be enhanced by using celecoxib, which targets COX-2, to enhance the sensitivity of lung cancer cells to NK cell-mediated cytotoxicity.

APX-115A, a pan-NADPH Oxidase Inhibitor, Induces Caspase-dependent Cell Death by Suppressing NOX4-ROS Signaling in EBV-infected Retinal Epithelial Cells.

PURPOSE: Epstein-Barr virus is a γ-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.

Combination treatment with erlotinib and ampelopsin overcomes erlotinib resistance in NSCLC cells via the Nox2-ROS-Bim pathway.

OBJECTIVES: Erlotinib, a tyrosine kinase inhibitor (TKI) of epidermal growth factor receptor (EGFR), has been shown to have a dramatic effect in non-small cell lung cancer (NSCLC) patients with EGFR mutation. However, the presence of primary resistance or acquired resistance to EGFR-TKI is the most common reason for switching to other anti-cancer agents. Even though there are newer agents that have activity in the presence of the T790M mutation, identification of potential agents that could overcome resistance to EGFR-TKI is still needed for the treatment of NSCLC patients. MATERIALS AND METHODS: In this study, we used erlotinib-resistant NSCLC cell lines to investigate the effects of combination treatment with erlotinib and ampelopsin. After treatment with either single or combination, cell viability and cell death were determined with WST-1 assay, trypan blue exclusion method, colony forming assay, annexin-V staining assay and western blot assay. The content of ROS was evaluated by FACS analysis using H2DCF-staining method. To determine the effect of Nox2 and Bim on the combined treatment with erlotinib and ampelopsin-induced cell death, we transfected with Nox2 or Bim specific siRNA and performed with western blot assay for evaluation of its expression. RESULTS: Combined treatment with erlotinib and ampelopsin at non-cytotoxic concentrations significantly induced caspase-dependent cell death in erlotinib-resistant NSCLC cells. Furthermore, cell death resulted in the accumulation of reactive oxygen species (ROS) through upregulation of nicotinamide adenine dinucleotide phosphate oxidase 2 (Nox2) expression, a direct source of ROS. The expression level of Bim increased with combination treatment, but not with either treatment alone. CONCLUSION: Here in this study, we demonstrate that the combination of erlotinib and ampelopsin induces cell death via the Nox2-ROS-Bim pathway, and ampelopsin could be used as a novel anti-cancer agent combined with EGFR-TKI to overcome resistance to erlotinib in EGFR-mutant NSCLC.