Screening and identification of emodin as an EBV DNase inhibitor to prevent its biological functions.

BACKGROUND: The Epstein-Barr virus (EBV) is a prevalent oncovirus associated with a variety of human illnesses. BGLF5, an EBV DNase with alkaline nuclease (AN) activity, plays important roles in the viral life cycle and progression of human malignancies and has been suggested as a possible diagnostic marker and target for cancer therapy. Methods used conventionally for the detection of AN activity, radioactivity-based nuclease activity assay and DNA digestion detection by gel electrophoresis, are not suitable for screening AN inhibitors; the former approach is unsafe, and the latter is complicated. In the present study, a fluorescence-based nuclease activity assay was used to screen several natural compounds and identify an EBV DNase inhibitor. RESULTS: Fluorescence-based nuclease activity assays, in which the DNA substrate is labelled with PicoGreen dye, are cheaper, safer, and easier to perform. Herein, the results of the fluorescence-based nuclease activity assay were consistent with the results of the two conventional methods. In addition, the PicoGreen-labelling method was applied for the biochemical characterisation of viral nucleases. Using this approach, we explored EBV DNase inhibitors. After several rounds of screening, emodin, an anthraquinone derivative, was found to possess significant anti-EBV DNase activity. We verified the efficacy of emodin using the conventional DNA-cleavage assay. Furthermore, using comet assay and micronucleus formation detection, we confirmed that emodin can inhibit DNase-induced DNA damage and genomic instability. Additionally, emodin treatment inhibited EBV production. CONCLUSIONS: Using a PicoGreen-mediated nuclease activity assay, we successfully demonstrated that emodin has the potential to inhibit EBV DNase nuclease activity. Emodin also inhibits EBV DNase-related biological functions, suggesting that it is a potential inhibitor of EBV DNase.

The Application of Emodin Treatment on Nasopharyngeal Carcinoma Therapy.

Nasopharyngeal carcinoma (NPC) is a malignancy prevailing in Taiwan, Hong Kong, Southern China, Southeast Asia, and North Africa. Although early-stage NPC responds well to the primary treatment of radio-chemotherapy, the mortality rate of advanced NPC remains high. Therefore, developing new therapies for nasopharyngeal carcinoma is an urgent task. Emodin is an anthraquinone derivative mainly found in Rheum palmatum. Emodin has been found to possess many anti-cancer functions against various types of cancers, but they are less discussed in the treatment of NPC. This review organized the different studies about the anti-NPC activity of emodin and discussed the potential and challenges of emodin treatment in NPC therapy.

Epstein-Barr Virus BRLF1 Induces PD-L1 Expression in Nasopharyngeal Carcinoma Cells.

Nasopharyngeal carcinoma (NPC) is a specific human malignancy with unique geographic distribution and genetic backgrounds. Although early treatment with radio-chemotherapy has been proven effective for NPC therapy, its therapeutic efficacy substantially diminishes in the late stages of this malignancy. In the tumor microenvironment of NPC, PD-L1 has been demonstrated as a critical factor in impairing T cell activation. As an etiological role for NPC development, it is found that Epstein-Barr virus (EBV) latent proteins upregulated PD-L1 expression. However, whether EBV lytic protein affects PD-L1 expression remains unclear. In this study, through monitoring the mRNA expression pattern of lytic genes and PD-L1 in EBV-positive NPC cell line NA, EBV immediately-early gene BRLF1(Rta) was found to have the potential for PD-L1 activation. Furthermore, we identified that Rta expression enhanced PD-L1 expression in mRNA and protein levels through quantitative real-time polymerase chain reaction and western blotting analysis. The luciferase reporter assay revealed that Rta expression enhanced PD-L1 promoter activity. We also demonstrated that Rta-induced PD-L1 expressions could impair interleukin 2 secretion of T cells, and this mechanism may be through ERK activation. These results displayed the importance of EBV Rta in PD-L1 expression in NPC and may give an alternative target for NPC therapy.

Ciliogenic RFX transcription factors regulate FGF1 gene promoter.

Fibroblast growth factor 1 (FGF1) has been shown to regulate cell proliferation, cell division, and neurogenesis. Human FGF1 gene 1B promoter (-540 to +31)-driven green fluorescence (F1BGFP) was shown to recapitulate endogenous FGF1 gene expression. It can also be used to isolate neural stem/progenitor cells (NSPCs) and glioblastoma stem cells (GBM-SCs) from developing mouse brains and human glioblastoma tissues, respectively. However, the regulatory mechanisms of FGF-1B promoter and F1BGFP(+) cells are not clear. In this study, we present several lines of evidence to show the roles of ciliogenic RFX transcription factors in the regulation of FGF-1B gene promoter and F1BGFP(+) cells: (i) RFX1, RFX2, and RFX3 transcription factors could directly bind the 18-bp cis-element (-484 to -467), and contribute to the regulation of FGF1 promoter and neurosphere formation. (ii) We demonstrated RFX2/RFX3 complex could only be detected in the nuclear extract of FGF-1B positive cells, but not in FGF-1B negative cells. (iii) Protein kinase C inhibitors, staurosporine and rottlerin, could decrease the percentage of F1BGFP(+) cells and their neurosphere formation efficiency through reducing the RFX2/3 complex. (iv) RNA interference knockdown of RFX2 could significantly reduce the percentage of F1BGFP(+) cells and their neurosphere formation efficiency whereas overexpression of RFX2 resulted in the opposite effects. Taken together, this study suggests ciliogenic RFX transcription factors regulate FGF-1B promoter activity and the maintenance of F1BGFP(+) NSPCs and GBM-SCs.

Establishing F1A-CreERT2 Mice to Trace Fgf1 Expression in Adult Mouse Cardiomyocytes.

Fibroblast growth factor 1 (FGF1) regulates many biological and physiological processes. In mice, Fgf1 gene contains at least three upstream promoters and are alternatively spliced to the first protein coding exon, giving rise to different Fgf1 mRNA variants (1A, 1B and 1G). Among them, the Fgf1A transcript is predominantly expressed in the heart. FGF1 can induce cardiomyocyte regeneration and cardiogenesis in vitro and in vivo. Here, we generated a novel mouse line using the Fgf1A promoter (F1A) driving the expression of the inducible Cre recombinase (CreERT2). We firstly demonstrated that the highest mRNA expression of CreERT2 were detected in the heart specifically of F1A-CreERT2 mice, similar to that of Fgf1A mRNA. The F1A-CreERT2 mice were crossed with ROSA26 mice, and the F1 mice were analyzed. The LacZ-positive signals were detected exclusively in the heart after tamoxifen administration. The CreERT2-mediated recombination in the tissues is monitored through LacZ-positive signals, indicating the in situ localization of F1A-positive cells. Consistently, these F1A-positive cells with RFP-positive signals or LacZ-positive blue signals were co-localized with cardiomyocytes expressing cardiac troponin T, suggesting cardiomyocyte-specific activation of Fgf1A promoter. Our data suggested that the F1A-CreERT2 mouse line could be used for time-dependent and lineage tracing of Fgf1A-expressing cells in vivo.

Isolation of neural stem/progenitor cells by using EGF/FGF1 and FGF1B promoter-driven green fluorescence from embryonic and adult mouse brains.

Fibroblast growth factor 1 (FGF1) and FGF2 have been shown to maintain the proliferation, self-renewal and multipotent capacities of neural stem/progenitor cells (NSPCs) in vitro. FGF1 is unique for binding to all known FGF receptors. In this study, we investigated if exogenous EGF and FGF1 could be used in the isolation of NSPCs from embryonic mouse brains. We demonstrated that EGF/FGF1-responsive cells exhibited lower proliferation rate and neurosphere formation efficiency than EGF/FGF2-responsive NSPCs. However, EGF/FGF1-responsive mouse brain cells exhibited better neural differentiation capacities than EGF/FGF2-responsive NSPCs at E11.5. Using F1BGFP reporter, we further demonstrated that F1BGFP+ cells showed similar multipotent capacities to CD133+ NSPCs, and could be induced more efficiently toward neuronal differentiation. Our results suggested that EGF/FGF1-responsive cells from E11.5 mouse brains could self-renew and have better multipotency than EGF/FGF2-responsive NSPCs. Further, CD133+ and F1BGFP+ NSPCs may also represent different subsets of NSPCs during neural development and adult neurogenesis.

Emodin Inhibits EBV Reactivation and Represses NPC Tumorigenesis.

Nasopharyngeal carcinoma (NPC) is a unique malignancy derived from the epithelium of the nasopharynx. Despite great advances in the development of radiotherapy and chemotherapy, relapse and metastasis in NPC patients remain major causes of mortality. Evidence accumulated over recent years indicates that Epstein-Barr virus (EBV) lytic replication plays an important role in the pathogenesis of NPC and inhibition of EBV reactivation is now being considered as a goal for the therapy of EBV-associated cancers. With this in mind, a panel of dietary compounds was screened and emodin was found to have potential anti-EBV activity. Through Western blotting, immunofluorescence, and flow cytometric analysis, we show that emodin inhibits the expression of EBV lytic proteins and blocks virion production in EBV- positive epithelial cell lines. In investigating the underlying mechanism, reporter assays indicated that emodin represses Zta promoter (Zp) and Rta promoter (Rp) activities, triggered by various inducers. Mapping of the Zp construct reveals that the SP1 binding region is important for emodin-triggered repression and emodin is shown to be able to inhibit SP1 expression, suggesting that it likely inhibits EBV reactivation by suppression of SP1 expression. Moreover, we also show that emodin inhibits the tumorigenic properties induced by repeated EBV reactivation, including micronucleus formation, cell proliferation, migration, and matrigel invasiveness. Emodin administration also represses the tumor growth in mice which is induced by EBV activation. Taken together, our results provide a potential chemopreventive agent in restricting EBV reactivation and NPC recurrence.

Inhibition of neurosphere formation in neural stem/progenitor cells by acrylamide.

Previous studies showed that transplantation of cultured neural stem/progenitor cells (NSPCs) could improve functional recovery for various neurological diseases. This study aims to develop a stem cell-based model for predictive toxicology of development in the neurological system after acrylamide exposure. Treatment of mouse (KT98/F1B-GFP) and human (U-1240 MG/F1B-GFP) NSPCs with 0.5 mM acrylamide resulted in the inhibition of neurosphere formation (definition of self-renewal ability in NSPCs), but not inhibition of cell proliferation. Apoptosis and differentiation of KT98 (a precursor of KT98/F1B-GFP) and KT98/F1B-GFP are not observed in acrylamide-treated neurospheres. Analysis of secondary neurosphere formation and differentiation of neurons and glia illustrated that acrylamide-treated KT98 and KT98/F1B-GFP neurospheres retain the NSPC properties, such as self-renewal and differentiation capacity. Correlation of acrylamide-inhibited neurosphere formation with cell-cell adhesion was observed in mouse NSPCs by live cell image analysis and the presence of acrylamide. Protein expression levels of cell adhesion molecules [neural cell adhesion molecule (NCAM) and N-cadherin] and extracellular signal-regulated kinases (ERK) in acrylamide-treated KT98/F1B-GFP and U-1240 MG/F1B-GFP neurospheres demonstrated that NCAM decreased and phospho-ERK (pERK) increased, whereas expression of N-cadherin remained unchanged. Analysis of AKT (protein kinase B, PKB)/ß-catenin pathway showed decrease in phospho-AKT (p-AKT) and cyclin D1 expression in acrylamide-treated neurospheres of KT98/F1B-GFP. Furthermore, PD98059, an ERK phosphorylation inhibitor, attenuated acrylamide-induced ERK phosphorylation, indicating that pERK contributed to the cell proliferation, but not in neurosphere formation in mouse NSPCs. Coimmunoprecipitation results of KT98/F1B-GFP cell lysates showed that the complex of NCAM and fibroblast growth factor receptor 1 (FGFR1) is present in the neurosphere, and the amount of this complex decreases after acrylamide treatment. Our results reveal that acrylamide inhibits neurosphere formation through the disruption of the neurosphere architecture in NSPCs. The downregulation of cell-cell adhesion resulted from decreasing the levels of NCAM as well as the formation of NCAM/FGFR complex.

Human FGF1 promoter is active in ependymal cells and dopaminergic neurons in the brains of F1B-GFP transgenic mice.

FGF1 is involved in multiple biological functions and exhibits the importance in neuroprotective effects. Our previous studies indicated that, in human brain and retina, the FGF1B promoter controlled the expression of FGF1. However, the exact function and regulation of FGF1 in brain is still unclear. Here, we generated F1B-GFP transgenic mice that expressed the GFP reporter gene under the control of human FGF1B promoter (-540 to +31). Using the fresh brain sections of F1B-GFP transgenic mice, we found that the F1B-GFP cells expressed strong fluorescent signals in the ventricular system throughout the brain. The results of immunohistochemistry further showed that two distinct populations of F1B-GFP(+) cells existed in the brains of F1B-GFP transgenic mice. We demonstrated that one population of F1B-GFP(+) cells was ependymal cells, which distributed along the entire ventricles, and the second population of F1B-GFP(+) cells was neuronal cells that projected their long processes into multiple directions in specific areas of the brain. The double labeling of F1B-GFP(+) cells and tyrosine hydroxylase indicated that a subpopulation of F1B-GFP(+) -neuronal cells was dopaminergic neurons. Importantly, these F1B-GFP(+) /TH(+) cells were distributed in the main dopaminergic neuronal groups including hypothalamus, ventral tegmental area, and raphe nuclei. These results suggested that human FGF1B promoter was active in ependymal cells, neurons, and a portion of dopaminergic neurons. Thus, the F1B-GFP transgenic mice provide an animal model not only for studying FGF1 gene expression in vivo but also for understanding the role of FGF1 contribution in neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease.

Using Drosophila eye as a model system to characterize the function of mars gene in cell-cycle regulation.

Human hepatoma up-regulated protein (HURP), a cell-cycle regulator, is found consistently overexpressed in human hepatocellular carcinoma. At present, the function of HURP in cell-cycle regulation and carcinogenesis remains unclear. In database mining, we have identified a mars gene in Drosophila, which encodes a protein with a high similarity to HURP in its guanylate kinase-associated protein (GKAP) motif. Overexpression but not down-regulation of mars in eye discs resulted in a higher mitotic index along with a high frequency of mitotic defects, including misalignment of chromosomes and mispositioned centrosomes, at the second mitotic wave (SMW). The consequence of mitotic defects impairs cell-cycle progression, and causes cell death posterior to the furrow. Immunocytochemical studies also have indicated that the expression of Mars is cell cycle regulated, and that its subcellular localization is dynamically changed during cell-cycle progression. Furthermore, we also demonstrated that the first 198 amino acids at the N-terminus of Mars are responsible for the degradation of Mars in non-mitotic cells. Together, we report the use Drosophila eye as a model system to characterize the function of the mars gene in cell-cycle regulation.