Interference of Fisetin with Targets of the Nuclear Factor-κB Signal Transduction Pathway Activated by Epstein-Barr Virus Encoded Latent Membrane Protein 1

The flavonoid fisetin (3, 3, 4, 7-tetrahydroxyflavone) is the main component of lacquer and is found in strawberries, apples, and grapes. The lacquer, lac tree extract, has been used as Chinese medicine in Asia for thousands of years. Several recent studies have demonstrated that the flavonoid has anticancer effects against several cancer types. Our preliminary data have indicated that fisetin induces cell cycle arrest and causes apoptosis in hepatocellular carcinoma cells (Li et al., 2010; 2011). However, the effect of fisetin on nasopharyngeal carcinoma (NPC) has not been studied extensively. Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) has been known to have oncogenic properties during latent infection in nasopharyngeal carcinoma (NPC) . In human epithelial cells, LMP1 alters many functional properties that are involved in tumor progression and invasions (Kim et al., 2000; Adham et al., 2012; Bambang et al., 2010). Activation of different signal transduction pathways mediates various downstream pathological effects of LMP1 expression, including cell proliferation, anti-apoptosis and metastasis (Eliopoulos et al, 2001; Guo et al., 2012). NF-κB participates in the induction of cellular genes encoding apoptosis molecules (Xu et al., 2012; Yang et al., 2012). In NPC cells, NF-κB plays a critical role in LMP1


Introduction
The flavonoid fisetin (3, 3, 4, 7-tetrahydroxyflavone) is the main component of lacquer and is found in strawberries, apples, and grapes.The lacquer, lac tree extract, has been used as Chinese medicine in Asia for thousands of years.Several recent studies have demonstrated that the flavonoid has anticancer effects against several cancer types.Our preliminary data have indicated that fisetin induces cell cycle arrest and causes apoptosis in hepatocellular carcinoma cells (Li et al., 2010;2011).However, the effect of fisetin on nasopharyngeal carcinoma (NPC) has not been studied extensively.
Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) has been known to have oncogenic properties during latent infection in nasopharyngeal carcinoma (NPC) .In human epithelial cells, LMP1 alters many functional properties that are involved in tumor progression and invasions (Kim et al., 2000;Adham et al., 2012;Bambang et al., 2010).Activation of different signal transduction pathways mediates various downstream pathological effects of LMP1 expression, including cell proliferation, anti-apoptosis and metastasis (Eliopoulos et al, 2001;Guo et al., 2012).

Cell culture and treatment
NPC cell lines CNE1 (well-differentiated) obtained from Institute of Virology, Chinese Academy of Preventive Medicine, CNE1-LMP1 was derived from CNE1 cells transfected with EBV LMP-1 eukaryotic expression plasmid PAT-GFP-LMP1, which was established previously in department of pathology.Cells were cultured in media with 10% FBS, 100U/mL penicillin and 0.1mg/ mL streptomycin at 37˚C with 5% CO 2 .CNE1-LMP1 cells were especially cultured in media contained 0.5µg/ mL puromycin.Cells were grown to 80% confluence, then digested with 0.25% trypsin and transferred into a new plate with fresh media.Fisetin dissolved in dimethyl sulfoxide (final concentration 0.1% vol/vol) was used for the treatment of cells.The cells (60-70% confluent) were treated with fisetin (6.25-100µM) for 24h in complete growth medium.

Cytotoxicity
For MTT, cells were plated at 8×10 3 cells per well in 200µl of complete culture medium containing 6.25-100µM concentration of fisetin in 96-well microtiter plates for 24h.After incubation for 24h at 37˚C in a humidified incubator, MTT [5mg/ml in phosphate-buffered saline (PBS)] was added to each well and incubated for 4h.The supernatant was removed, the pellet was resuspended and 150µl DMSO was added to each well, followed by agitation at room temperature for 15 min to dissolve the precipitates.The absorbance (A) value was measured at 490 nm.The inhibition ratio was calculated as: the inhibition ratio of cell growth= (1-value A of each sample/ value A of control)×100%.

Immunofluorescent staining
Cover slips were seeded with CNE1 and CNE-LMP1 cells, and the cells were treat with fisetin (0-100µM) for 24h.The slips were washed with PBS, fixed with 1:1 acetone-methanol, cells were incubated overnight with the primary antibodies (NF-κB p65, 1:100) at 4℃, the antigenic sites were localized using FITC-conjugated rabbit anti-goat IgG (1:100).Images of the antigenic sites were captured by a laser scanning confocal microscope.

Transfection
Transfections were done using LipofectAMINE 2000Transfection Reagent.Cells seeded at 24-well culture plates were cultured in RPMI-1640 supplemented with 10% FBS at 37℃ for 24h.Briefly, 2µl of lipofectamine and 0.8µg of DNA were diluted in 500µl of RPMI-1640 followed by equilibration at room temperature for 5 min after mixing.The lipofectamine-DNA complex was added to cells and incubated for 6h.Cells were then washed with PBS and replenished with RPMI-1640 containing 20% serum.At 6h after transfection, the cells were incubated with 0, 25, 50, 100µM of fisetin for 24h.

Luciferase assays
Cells were co-transfected with expression vectors NF-κB promoter reporters, and the internal control plasmid TK-RL for 6h, cells were treated with fisetin for 24h and then harvested by lysis with 1×Passive Lysis Buffer.Lysate was added to each well of a non-transparent 96well plate and mixed with luciferin.The luciferase activity was measured with a BioTek ELISA reader, followed by measurement of the firefly luciferase activity.Luciferase activity was then quenched with GLO Stop reagent, and new luciferin was added.The ratios of the two readings were calculated and interpreted as the calibrated reporter activity of the transcription gene.The data were presented as mean±standard deviation (SD), and were derived from at least three independent experiments.

Western blot analysis
Following the treatment of CNE1 and CNE1-LMP1 cells with fisetin (0, 50µM, 24h), the media was aspirated, the cells were washed twice with ice-cold PBS and lysed in cold lysis buffer [50 mM Tris-HCl, pH 7.5, 150 mM sodium chloride, 0.5% a-cholate, 0.1% SDS, 2mM EDTA, 1% Triton X-100, and 10% glycerol], Lysates were incubated for 20 min on ice and centrifuged at 12000g for 15 min.The supernatant was collected.After the treatment of cells with fisetin, nuclear and cytoplasmic fractions of cells were extracted and the protein concentration was determined by BCA assay reagent.The protein was electrophoresed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes.The membranes were blocked with 50g/l non-fat dried milk in TBST (TBS, 0.5ml/l Tween-20) for 1h at room temperature and incubated overnight at 4˚C with the first anti-body against human NF-κB (1:500) IκBα (1:500), cyclinD1 (1:1000) and a phosphorylated antibody IκBα (1:500), followed by incubation with an HRP-conjugated secondary antibody at room temperature for 1h.Enhanced chemiluminescence (ECL) was used to detect the results.
Fisetin inhibited the expression and translocation of NF-κB in CNE1-LMP1 cells.It is well known that NF-κB transcription factor is associated with cellular transformation and has been identified to be upregulated in nasopharyngeal carcinoma.In western blot analysis, we observed decreased level of p65 subunit of NF-κB in cytoplasm and concomitant decrease in p65 subunit in nuclear lysates of CNE1-LMP1 treated with fisetin, but there was no apparent change for CNE1 cells (Figure 3A, 3D).To confirm the results, we labeled fisetin-treated cells with p65 -specific antibodies.As shown, the CNE1-LMP1 cells that transfected with an EBV-LMP 1 overexpression plasmid displayed the stronger p65 expression.As expected, translocation of p65 was also inhibited by fisetin.The expression of p65 was downregulated and the translocation of the proteins to the nucleus were inhibited by fisetin as shown by diminished green fluorescence in p65 pane l (Figure 4).
Fisetin inhibited the expression of phosphorylation and degradation of IκBα.A crucial step in the activation of NF-κB is the phosphorylation of IκBα by IκBα kinase complex.These results indicated that fisetin treatment of CNE1-LMP1 cells resulted in inhibition of phosphorylation and degradation of IκBα, but there was no apparent change for CNE1 cells (Figure 3B, 3E).
Fisetin reduced the expression of cyclin D1.Cyclin D1 is frequently over-expressed in nasopharyngeal carcinoma and its increased expression is known to contribute to the abnormal growth and tumorigenicity.In our western blot analysis, we observed diminished levels of cyclin D1 in CNE1-LMP1, but there was no apparent change for CNE1 cells (Figure 3C, 3F).

Discussion
Many flavonoids possess anti-tumor or growth suppressive capacity against various human tumor cell lines, which lead to arrest the tumor promotion and progression, presumably by affecting or disturbing crucial factors that control cell proliferation, invasion, or apoptosis.Previous studies have demonstrated that fisetin had extensively anti-tumorigenic ability in various cancer cells.Therefore, fisetin may be one of its critical features as a chemopreventive agent.In this study, we have investigated the effects of fisetin on CNE1, CNE1-LMP1.CNE-1 showed no LMP1 expression, CNE1-LMP1 that established from CNE-1 transfected with a LMP-1 overexpression plasmid (Chen, 2002), displayed a very strong LMP1 expression.We demonstrated that fisetin suppressed the growth of CNE1 and CNE1-LMP1 cells in vitro.The results of the screening trend to show that carcinoma cells, especially CNE1-LMP1are more sensitive to fisetin than CNE1.
Latent membrane protein-1 (LMP-1) is EBV-encoded oncoprotein that could promote NPC to progress via activation of multiple signaling (Zheng et al., 2007;Yoshizaki, 2002).LMP1, a multifunctional oncoprotein, is a powerful activator of the transcription factor NF-κB.In NPC, LMP1 phosphorylates IκBα, thus activating NF-κB through two pathways: the TRAFs pathway and the TRADD (tumor necrosis factor receptor associated death domain protein) pathway.Active NF-κB induces the cell immortalization via the upregulation of the telomerase activity through the translocation of hTERT protein bound to NF-κB (Zuo et al., 2011), and promotes the cell proliferation via regulating survivin, CyclinD1, CyclinE, and EGFR signaling, etc (Zuercher et al., 2012;Ramdass et al., 2007).More and more evidence had demonstrated that NF-κB activation by LMP1 play a pivotal role in the proliferation and migration of NPC cells and targeting NF-κB may be a potential interest strategy in NPC treatments.We demonstrated that fisetin could block the NF-κB activities triggered by LMP1.Nuclear translocation of NF-κB (p65) from the cytoplasm into the nucleus induced by LMP1 can be prevented by fisetin.Furthermore, NF-κB was accumulated within the nuclear compartment if not treated with fisetin, but was translocated into the cytosolic compartment after fisetin treatment.Fisetin also inhibited IκBα phosphorylation and cyclin D1 protein expression, These results suggested that fisetin interferes the NF-κB signal transduction pathways triggered by LMP1.However, the manner in which NF-κB pathway was modified and its stability regulated has yet to be elucidated.

Figure 1 .Figure 2 .Figure 3 .
Figure 1.Effects of Fisetin Treatment on Cell Proliferation in CNE1 and CNE-LMP1 Cells.CNE1 and CNE-LMP1cells were treated with fisetin (6.25-100µM) for 24 and the inhibitory rate of cells were determined by MTT assay.The data are expressed as the percentage of cell inhibitory rate and represent the means±SD of three experiments in which each treatment was performed in multiple wells.*Indicatesp<0.05