Inhibition of Epstein-Barr virus reactivation by the flavonoid apigenin
Wu et al. Journal of Biomedical Science
Inhibition of Epstein-Barr virus reactivation by the flavonoid apigenin
Chung-Chun Wu 0
Chih-Yeu Fang 0 2
Yu-Jhen Cheng 0
Hui-Yu Hsu 0
Sheng-Ping Chou 0
Sheng-Yen Huang 0
Ching-Hwa Tsai 1
Jen-Yang Chen 0 1
0 National Institute of Cancer Research, National Health Research Institutes , No.35, Keyan Road, Zhunan Miaoli, Miaoli County , Taiwan
1 Department of Microbiology, College of Medicine National Health Research Institutes, National Taiwan University , No.35, Keyan Road, Zhunan Town, Miaoli County, Taipei , Taiwan
2 Department of Pathology, Wan Fang Hospital, Taipei Medical University , Taipei 116 , Taiwan
Background: Lytic reactivation of EBV has been reported to play an important role in human diseases, including NPC carcinogenesis. Inhibition of EBV reactivation is considered to be of great benefit in the treatment of virus-associated diseases. For this purpose, we screened for inhibitory compounds and found that apigenin, a flavonoid, seemed to have the ability to inhibit EBV reactivation. Methods: We performed western blotting, immunofluorescence and luciferase analyses to determine whether apigenin has anti-EBV activity. Results: Apigenin inhibited expression of the EBV lytic proteins, Zta, Rta, EAD and DNase in epithelial and B cells. It also reduced the number of EBV-reactivating cells detectable by immunofluorescence analysis. In addition, apigenin has been found to reduce dramatically the production of EBV virions. Luciferase reporter analysis was performed to determine the mechanism by which apigenin inhibits EBV reactivation: apigenin suppressed the activity of the immediate-early (IE) gene Zta and Rta promoters, suggesting it can block initiation of the EBV lytic cycle. Conclusion: Taken together, apigenin inhibits EBV reactivation by suppressing the promoter activities of two viral IE genes, suggesting apigenin is a potential dietary compound for prevention of EBV reactivation.
Epstein-Barr virus; Apigenin; Reactivation; Nasopharyngeal carcinoma
Epstein-Barr virus, a member of the γ-herpesviruses,
infects most of the human population worldwide . It
plays a causative role in infectious mononucleosis, hairy
leukoplakia, and post-transplant lymphoproliferative
disorder  and is highly associated with several human
malignancies, including Burkitt’s lymphoma (BL) and
nasopharyngeal carcinoma (NPC). EBV mainly infects
human circulating B cells and is maintained in a latent
state. Upon stimulation by chemical agents, e.g.
12-otetradecanoyl-phorbol-1,3-acetate (TPA) and sodium
butyrate (SB), human IgG or cytokines, EBV enters the
lytic stage. It sequentially expresses immediate early (IE),
early (E) and late (L) proteins and, eventually, mature
virions are released .
In the recent decade, increasing evidence has
suggested that EBV lytic reactivation plays an important
role in various human malignancies. In
seroepidemiological studies, elevation of antibody titers against EBV
lytic proteins in NPC and BL patients has suggested that
EBV reactivation is highly correlated with cancer
progression, poor prognosis and tumor recurrence of NPC
[2–4]. Retrospective studies revealed that NPC patients
have elevated antibody titers against EBV lytic antigens
prior to diagnosis and prospective surveys have revealed
that individuals with elevated antibody titers have a
higher incidence of NPC [5–7]. Moreover, the proteins
and mRNAs of EBV lytic genes were detectable in
clinical samples from NPC patients [8–10]. Recently, we
found that EBV reactivation induces genomic instability
and enhances tumor progression [11, 12]. EBV lytic
proteins, such as viral DNase, terminase and kinase, also
have been shown to have the ability to induce genomic
instability via different mechanisms [13–15]. These
reports revealed that inhibition of EBV reactivation is
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beneficial for cancer prevention and therapy [16, 17].
Several types of compounds also have been developed
for the inhibition of EBV reactivation: (i) Nucleoside
analogs, which inhibit the EBV lytic cycle by blocking
DNA replication, are used extensively in antiviral
therapy (e.g. acyclovir, ACV, and ganciclovir, GCV) . (ii)
Non-nucleoside drugs have been developed to block
EBV replication (e.g. maribavir) . (iii) Dietary
ingredients, e.g. retinoic acid, epigallocatechin gallate,
curcumin and sulforaphane, also have been suggested to have
the potential to inhibit the EBV lytic cycle [20–23].
Regarding clinical application, dietary compounds are
attractive for the inhibition of EBV reactivation because
of their safety and convenience. We screened several
dietary compounds to identify those are able to inhibit
the EBV lytic cycle and found that apigenin has the
ability to inhibit the EBV lytic induction effectively.
Apigenin is a member of the flavonoids, which are
present in abundance in common fruits and vegetables
. Apigenin has anti-oxidative, anti-mutagenic,
anticarcinogenic, anti-inflammatory, anti-proliferative and
anti-progressional properties . However, the
association between these biological functions and, the
antiviral effect of apigenin is less well understood.
In this study, we found apigenin inhibits EBV
reactivation into the lytic cycle and virion production by
EBVpositive NPC cells. Moreover, we addressed the question
whether apigenin represses the promoter activities of the
EBV IE genes, Zta and Rta, to explore the possible
mechanism of this inhibitory effect. This study gives
new insight into the biological application of apigenin
and provides an alternative choice for anti-EBV therapy.
Compounds and antibodies
Apigenin and the induction agents, TPA, SB, TSA,
SAHA and romidepsin were purchased from
SigmaAldrich Co. Antibodies used in western blotting and
immunofluorescence analysis include anti-EBV Rta
467 (unpublished), anti-BMRF1 (EAD) 88A9 ,
anti-EBV Zta 4 F10, anti-DNase 311H , and
antiβ-actin (Sigma-Aldrich Co.).
NA and HA cells, are EBV converted cells obtained by
co-culture of rAkata cells with TW01 and HONE-1 cells,
respectively, and were selected by G418 (Sigma-Aldrich
Co) treatment . All epithelial cell lines were
maintained in DMEM (Dulbecco’s modified Eagle’s medium)
supplemented with 10% fetal calf serum (FCS). P3HR1
, an EBV-positive Burkitt’s lymphoma cell line, was
maintained in RPMI-1640 supplemented with 10% FCS.
The cytotoxicity of apigenin to each cell line was
determined by WST-1 assay (Invitrogen) according to the
manufacturer’s instructions. The half maximum
cytotoxic concentration (CC50) for each cell line was the
concentration of apigenin which killed 50% of the cells.
The results were averaged from at least three
independent experiments to calculate the mean and standard
Western blotting analysis
Western blotting has been described in our previous
report . Briefly, the samples were subjected to
SDSPAGE and then transferred to Hybond-C membranes
(Amersham Biosciences Ltd.). After blocking for 1 h, the
membranes were incubated with the antibodies indicated
for 24 h at room temperature and then washed three
times with washing buffer (10 mM Tris–HCl, pH 8.0,
0.9% NaCl). The blots were then treated with
horseradish peroxidase-labelled goat anti-mouse IgG (Amersham
Biosciences Ltd., diluted 1:20,000) for 1 h at room
temperature. After washing three times, the blots were
developed with freshly prepared substrate (Amersham
Biosciences Ltd.). The luminescence was detected by a
short exposure to x-ray film.
The cells indicated were seeded on cover slides for 24 h
and then treated with various concentrations of apigenin
for 25 h, or pretreated with apigenin for 1 h followed by
TPA (40 ng/ml) and SB (3 mM) treatment for a further
24 h. The cells then fixed with 2% formaldehyde for
10 min and permeabilized with 0.4% Triton X-100 in
PBS for a further 5 min. After washing three times, the
cells were blocked in 4% FCS in PBS for 30 min. The
cells were treated with anti-EAD antibody which was
diluted in 1:10 at 37 °C for 1 h. After washing,
rhodamine-conjugated goat anti-mouse IgG, diluted
1:100 in 4% FCS-PBS was added. After incubation with
secondary antibody at 37 °C for 1 h, the cells were
washed and observed by fluorescence microscopy. The
nuclei were visualized by DAPI (Sigma-Aldrich Co)
Determination of the copy number of the viral genome
The procedures for determination of viral genome copy
numbers were derived from a published paper .
Briefly, EBV-positive NPC NA cells (1 × 106 cells/well)
were incubated with TPA (40 ng/ml) and SB (3 mM) for
48 h after pre-treatment of apigenin for 1 h. The
supernatants were harvested and filtered through a 0.45 μM
filter, then each sample was incubated with DNase I and
10× DNase I buffer (10 mM Tris–HCl, 2.5 mM MgCl2,
0.5 mM CaCl2,and pH 7.6) at 37 °C for 60 min, then
2 mM EDTA (pH 8.0) was added to inhibit DNase I
activity. Each sample was then treated with 0.1 mg/ml
proteinase K (1:1 [vol/vol]) at 50 °C for 60 min and the
reactions were stopped at 75 °C for 20 min.
Subsequently, each sample and standards (1 μl, see the
description below) were examined for the BALF5
sequence (sense: 5′-CGGAGTTGTTATCAAAGAGGC-3′;
antisense: 5′-CGAGAAAGACGGAGATGGC-3′), the
DNA polymerase of EBV, by real-time quantitative PCR
(qPCR) amplification . The qPCR conditions were:
5 s denaturation at 95 °C, 20 s annealing at 60 °C and
2 s extension of primers at 72 °C for 45 cycles. The
specificity of the PCR reaction was monitored by
melting curve analysis (65–95 °C, 0.1 °C/s) in the
LightCycler 480 (Roche Applied Science). The results from
three independent experiments were used to calculate
the mean and standard deviation.
The 5′-serial-deleted mutants of Zp were reported
previously . The mutants of each domain of Zp were
constructed by means of PCR-based site-directed
mutagenesis. The primers used referred to the following
reports: mZIIIA (forward 5′-TAGAAACTATGCAG
AATTCACAGGCATTGCTAA) ; mZIIIB (forward
mZ1D-1 (forward 5′-ATGTACCTCATAGACAATACT
AAATTTAGCACGTC) ; mZ1D-2 (forward
5′-TCATAGACACACCGCCATTTAGCACGTCC) ; mZII-1
5′-CACGTCCCAAACGAATTCATCACAGAGGA) ; ZII-2 (forward 5′-CAAACCATGAC
Transfection and luciferase reporter activity analysis
The construction of the Zp and Rp reporter plasmids
was described in a previous report . Transfection
procedure was carried out using Lipofectamine 2000
(Invitrogen) according to the manufacturer’s
instructions. For Zp or Rp activation by TPA + SB, plasmid Zp
or Rp was first transfected into NA and parental TW01
cells. After 3 h transfection, apigenin was added or not
for pre-treatment for 1 h, and then TPA (40 ng/ml) and
SB (3 mM) were added to induce EBV into the lytic
cycle. For transfection of Zta-expressing plasmid plus Zp
or Rp, Rta-expression plasmid plus Zp or Rp, cells (2 ×
105 cells/well) were seeded 24 h before transfection.
Plasmid mixtures were transfected using the procedures
described above. After induction for 24 h, cells were
lysed in HEPES lysis solution and the lysates were
subjected to luciferase activity assay (Promega). Each
lysate sample was quantified for the expression of
β-actin to control for variation in the amount of each
sample (data not shown). For analysis of the activities of
the Zp mutants, by comparison with mock treatment
(M) of the vector control (V), for which the activity was
set to 1, the relative activities are indicated as N fold
induction over the activity of the vector control. The
fold of inhibition is the induction folds of the TPA + SB
plus apigenin (TS + A20 and TS + A50) groups
compared to that of the TPA + SB (TS) group. The mean
and standard deviation of each sample were calculated
from at least two independent experiments in duplicate.
The cytotoxicity of apigenin to epithelial cells
Apigenin, which is known as 4’, 5, 7,-trihydroxyflavone
(Figure 1a), is a flavonoid of the flavone structural type.
In order to rule out the interference of toxic effects of
apigenin treatment of the cells, we first tried to
determine the cytotoxicity of apigenin to NPC cells. Two
EBV-positive cell lines, NA and HA, were tested by
WST-1 assay for susceptibility to apigenin cytotoxicity
after 24 h and 48 h treatments. As shown in Figure 1b,
apigenin had almost no effect on NA cells after 24 h,
and had low cytotoxicity after 48 h. To the other NPC
cells line, HA, apigenin also showed little cytotoxicity
after 24 h, however, it had significant cytotoxicity with
20 ~ 100 μM for 48 h (Figure 1c). The cytotoxicity of
apigenin to the B cell line P3HR1 was also determined
(Figure 1d). To evaluate these data more precisely, the
values of half maximum of cytotoxicity concentration 50
(CC50) were calculated and are shown at the top of
Figures 1b to d. The CC50 values of all three cell lines for
24 h were greater than 200 μM, while the values for
48 h treatment of NA, HA and P3HR1 were 148, 69 and
158 μM (Figures 1b to d), respectively. To determine the
cytotoxicity of the combination of apigenin and TPA +
SB, NA, HA and P3HR1 cell lines were pre-treated with
various concentrations of apigenin for 1 h and then TPA
+ SB were added. After 24 and 48 h incubation, the cells
were subjected to WST-1 assay. The results indicated
that apigenin combined with TPA + SB treatment did
not cause severe cytotoxicity to the three cell lines
(Additional file 1). Taken together, we determined that
the CC50 values of apigenin are 200 to 295 μM and 69
to 158 μM for 24 and 48 h, respectively, which is similar
to the effect on other cancer cells . Thus we chose 1
~ 50 μM as our working concentrations for further
Inhibition of expression of the EBV lytic proteins by
We next sought to determine whether apigenin has the
ability to induce or inhibit EBV reactivation. Two EBV
positive-NPC cell lines, NA and HA, were used for these
experiments. After EBV reactivation, the
immediateearly genes Zta and Rta are expressed, followed by
numerous early and late proteins and, subsequently, the
Fig. 1 The cytotoxicity of apigenin to epithelial cells. (a) The chemical structure of apigenin. (b) NA, (c) HA and (d) P3HR1 cell lines were treated
with apigenin for 24 h. Cell viability was determined by WST-1 assay, as described in Methods. The values are means ± SD from at least two
separate experiments. CC50 values also were calculated and are given at the top of each group
release of infectious virions. To determine whether
apigenin can induce EBV reactivation, NA cells were
treated with apigenin for 24 h and cell lysates were
collected for the detection of EBV lytic proteins Zta, Rta,
EAD and DNase by western blot analysis. As shown in
the left panel of Figure 2a, apigenin did not induce any
lytic protein expression in NA cells, suggesting apigenin
cannot induce EBV reactivation. Next, we tried to
determine whether apigenin inhibits EBV reactivation in NPC
cells. For NPC cells, TPA + SB treatment is an effective
way to activate EBV into the lytic stage and induce lytic
protein expression . Moreover, to achieve greater
efficiency of apigenin treatment, plated cells were
pretreated with apigenin for 1 h prior to treatment with
TPA (40 ng/ml) and SB (3 mM). After a further 24 h
incubation, cell extracts were collected for detection of
lytic proteins by western blot analysis. For the NA cells,
the result showed that EBV could express lytic proteins
normally, however, protein expression was gradually
repressed following apigenin treatment, showing that
apigenin has the ability to inhibit EBV reactivation (right
panel, Figure 2a). To avoid the possibility of cell
specificity, we used another EBV-positive NPC cell line, HA, to
determine whether EBV reactivation can be inhibited by
apigenin treatment. The result shown in Figure 2b
reveals that administration of apigenin inhibited EBV
lytic reactivation with a dose-dependent tendency in HA
cells. The expression of lytic proteins was almost
completely blocked with 20 and 50 μM apigenin treatment,
suggesting a significant inhibitory effect (Figure 2b). In
addition, because B cells constitute another natural host
cell type of EBV, we investigated whether apigenin can
inhibit EBV reactivation in B cells. The EBV-positive
Burkitts’ lymphoma cell line P3HR1 was tested using the
procedure described above. As shown in Figure 2c,
apigenin treatment also inhibited EBV lytic protein in
P3HR1 cells. These results revealed that apigenin has
the capability to inhibit EBV lytic protein expression, no
matter whether in epithelial or B cells.
Immunofluorescence analysis of inhibition of the EBV
lytic cycle by apigenin treatment
Next, to confirm the inhibitory effect of apigenin, we
examined the induction or inhibition of EBV lytic reactivation by
detecting EAD expression using immunofluorescence
analysis without or with TPA + SB induction. NA and HA cells
Fig. 2 Apigenin inhibits the expression of EBV lytic proteins in EBV-positive Cells. (a) Epithelial NA cells were tested to detect the expression of
EBV lytic proteins. Left panel: for detection of enhancement of reactivation, various concentrations of apigenin were added to the cells for 25 h,
and then cell lysates were collected for western blotting. Right panel: for detection of inhibition of reactivation, the cells were pre-treated with
apigenin for 1 h, then TPA + SB co-treatment was used for EBV induction. After 24 h of incubation, the cell lysates were analyzed by western
blotting with antibodies against EBV Zta, Rta, EAD, DNase and β-actin. (b) Epithelial HA cells and (c) Burkitt’s lymphoma P3HR1 cells were tested
for detection of inhibition of EBV reactivation using a similar procedure
were incubated with various concentrations of apigenin for
25 h. The results were that apigenin cannot induce EAD
expression in NA and HA cells (Figure 3a and b, upper
panels). For detection of inhibition of EBV reactivation, NA
and HA cells were pre-treated with various concentrations
of apigenin for 1 h, then EBV was activated by adding TPA
+ SB. After a further 24 h incubation, the
immunofluorescence results revealed that the number of EAD-expressing
cells decreased gradually with treatment of NA cells with
increasing concentrations of apigenin (Figure 3a, lower
panel). Apigenin repressed the numbers of EAD-expressing
NA cells significantly at 10 μM, and blocked EAD
expression completely at 20 ~ 50 μM. A similar result was
observed for HA cells (Figure 3b, lower panel). Apigenin
inhibited the numbers of EAD-expressing HA cells
gradually in a dose-dependent manner (Figure 3b, lower
panel). To analyze this inhibitory effect in more detail, we
quantified the numbers of cells expressing EAD. As shown
in Figure 3c, the numbers of EAD-expressing cells
decreased gradually with apigenin treatment (Figure 3c).
Taken together, the results of western blotting and
immunofluorescence analysis, we conclude that apigenin
can repress EBV lytic protein expressions, suggesting it has
the ability to inhibit EBV reactivation.
Inhibition of EBV virion production by apigenin
We have already shown that apigenin blocks the
expression of EBV lytic proteins. A further question was
whether apigenin can inhibit the EBV lytic cycle
completely. NA cells were pre-treated with apigenin for
1 h, then TPA + SB was added. After incubation for
48 h, the supernatants were collected to measure the
amounts of EBV virions. As shown in Figure 4, the
production of EBV virions decreased gradually with
increasing apigenin treatment. EBV virion production
decreased significantly following treatment with 10 μM
apigenin and was eliminated almost completely with 20
and 50 μM apigenin after induction (Figure 4). This
result suggests that apigenin can inhibit not only EBV
lytic protein expression but also virion production.
Fig. 3 (See legend on next page.)
(See figure on previous page.)
Fig. 3 Apigenin decreases the populations of EAD-expressing cells, detected by immunofluorescence analysis NA (a) and HA (b) cells were
processed for immunofluorescence analysis (IFA). For detection of inhibition of reactivation, the cells were pre-treated with various
concentrations of apigenin for 1 h, then TPA + SB were added for EBV induction. After 24 h of incubation, the cells were analyzed by IFA with
antibody against EBV EAD. (c) The percentages of EAD-expressing cells in each sample were calculated. The values are means ± SD from three
Apigenin represses Zp and Rp activities following TPA +
SB treatment and Zta/Rta induction
Zta and Rta are the two most important proteins that
initiate the EBV lytic stage. To investigate further the
inhibitory mechanism of action of apigenin, we sought
to determine whether apigenin affects Zta and Rta
promoter activities (Zp and Rp) under different stimuli. For
chemical induction, luciferase reporters containing Zp
and Rp were transfected into NA cells for 3 h. The cells
were pre-treated with various amounts of apigenin for
1 h, TPA + SB were added for a further 24 h to induce
EBV reactivation. Luciferase activity was determined
subsequently as described in Materials and Methods. As
expected, for the positive control, the luciferase activities
of Zp and Rp both increased significantly after TPA + SB
induction, compared to the mock-transfected control
(Figure 5a, 0 μM). Meanwhile, the luciferase activities of
Zp and Rp were gradually repressed by increasing
concentrations of apigenin (Figure 5a). Co-treatment with
TPA + SB and 50 μM apigenin reduced the activities of
Zp and Rp to the mock-transfected control level. The
result of NA cells transfected with the empty vector PGL2
showed that all values were at background levels
In addition to inhibiting the induction by chemicals,
we tried to use another method of EBV induction to
elucidate further the possible mechanism by which apigenin
inhibits the EBV lytic cycle. Because the Zta response
element (ZRE) and Rta response element (RRE) are
Fig. 4 Apigenin inhibits virion production. After EBV reactivation,
culture media containing released EBV particles were collected for
qPCR analysis to detect the amount of EBV DNA in released EBV
particles, as described in Methods. The relative EBV copy numbers
were calculated. These are expressed as the relative folds to mock
control. The values are means ± SD from three separate experiments
present within Zp and Rp, respectively, Zp and Rp have
been reported to be transactivated by the Zta and Rta
proteins . Therefore, Zta- and Rta-expressing
plasmids were cotransfected with Zp or Rp into NA cells
for 3 h, followed by apigenin treatment for 24 h. As
expected, Zta expression in NA cells induced the
promoter activities of Zp and Rp without apigenin
treatment (Figure 5b, Zp & Rp, 0 μM). After co-treatment
with apigenin, the promoter activities of Zp and Rp were
reduced in a dose-dependent manner (Figure 5b, Zp &
Rp). In addition, Rta expression also induced Zp and Rp
activities, however, these were repressed by apigenin
treatment, compared to that of the control reporter
PGL2 (Figure 5c). In addition, more inducers were tested
to confirm these phenomena. The results indicated that
apigenin also inhibits Zp activity in a dose-dependent
manner following induction by various HDAC inhibitors
[35, 36] (Additional file 2).
To avoid the effect of EBV activity in NA cells, we
used the parental TW01 cells to detect the inhibitory
effects of apigenin, in a similar manner to that described
above. As shown in Figure 5d, TPA + SB treatment
induced Zp and Rp significantly and this increase was
inhibited by addition of apigenin. Similarly, increased Zp
and Rp activities induced by Zta and Rta expression also
were repressed by apigenin treatment of TW01 cells
(Figures 5e and f ).
Taken together, these results indicated that apigenin
inhibits the EBV lytic cycle initiation by repressing IE
promoter activities, stimulated by various inducers.
The ZIIIA/B, Z1D and ZII elements of Zp are important in
inhibition of apigenin
Because Zta is the first gene expressed after initiation of
the EBV lytic cycle, we tried to determine what elements
of Zp are essential for apigenin inhibition. For this
purpose, we mapped the response domains of Zp required
for apigenin inhibition. A series of 5’-deletion Zp
constructs spanning from −221 to +12 region of Zp was
transfected into TW01 cells for 3 h (Figure 6a, left
panel). The cells were then pre-treated with 20 and
50 μM apigenin for 1 h, then TPA + SB was added. After
treatment with TPA + SB for a further 24 h, the cell
extracts were collected subsequently for detection of
luciferase activity. For the TPA + SB - treated group
(TS), the activities of serial deletion constructs from
−221 to −134 were not affected, while deletion construct
Fig. 5 Apigenin represses Zp and Rp activities under different inductions. (a) Control plasmid PGL2, Zp, or Rp was transfected into NA cells. After
3 ~ 4 h of transfection, apigenin was added or not for pre-treatment for 1 h, and then TPA + SB were used to induce EBV into the lytic cycle. After
induction for a further 24 h, lysates were collected for measurement of luciferase activity. Zp or Rp were co-transfected with (b) Zta-expressing
and (c) Rta-expressing plasmids into NA cells for 3 h, then apigenin was added. After 24 h of transfection, luciferase activity was detected in the
cell lysates. (d) Zp or Rp plasmid was transfected into TW01 cells. After 3 ~ 4 h of transfection, apigenin was added or not for pre-treatment for
1 h, and then TPA + SB was used to induce EBV reactivation. After induction for 24 h, cells lysates were produced for measurement of luciferase
activity. Zp or Rp were co-transfected into TW01 cells with (e) Zta-expressing or (f) Rta-expressing plasmids. After 24 h of transfection, luciferase
activity was detected in the cell lysates. The mean and standard deviation of each sample were calculated based on duplicates from three
Zp-99 abolished a half of the Zp-221 activity, moreover,
Zp-80 and Zp-51 retained little activity compared to
vector control with mock treatment (Figure 6a, right panel).
In addition, treatment with 20 and 50 μM apigenin
repressed Zp activity in constructs Zp-221 to Zp-134
(Figure 6a, right panel), which showed a similar inhibition
fold compared to that of each individual TS group
(Figure 6a, right panel). However, the inhibition was
gradually compromised in the Zp-99 to Zp-51 constructs
(Figure 6a, right panel), suggesting the responsive
element for apigenin inhibition of Zp induction is within
the region −134 to −51.
There are three major domains located within the
region −134 to −51, ZIIIA/B, Z1D and ZII (Figure 6a, left
panel). ZIIIA/B is a regulatory region of Zp containing
several Zta-binding-elements (ZREs). Z1D is an
important domain involving Sp1/Sp3 and MEF2D regulation.
The ZII region has been found to be an essential
element for Zp activation and contains several transcriptional
factor binding sites, including ATF-1, ATF-2 and CREB.
Fig. 6 Identification of the response element of Zp required for apigenin inhibition of EBV. (a) A schematic diagram of the −221 to +1 region of
Zp that drives the luciferase gene in the reporter plasmid (left panel). For the analysis of activities of Zp deletion mutants in response to apigenin
plus TPA + SB induction. TW01 cells were transfected with these deletion mutants. Three hours after transfection, the cells were pre-treated with
apigenin for 1 h and TPA + SB were added to activate EBV (right panel). The relative luciferase activities were calculated as described in Methods.
(b) A schematic diagram of the indicated domain structure and cellular factor binding sites on Zp (−221 to +1). The relevant mutated sites are
indicated by black triangle (▲) on the diagram (upper panel). For analysis of luciferase activities of Zp mutants upon apigenin plus TPA + SB
treatment, TW01 cells were transfected with the mutants indicated for 3 h and pre-treated with apigenin, then TPA + SB were added for 24 h to
activate EBV (lower panel). The luciferase activities were detected and were calculated as described in Methods. The mean and standard deviation
of each sample were calculated in duplicate from at least two independent experiments
To determine which regulatory factor was important for
apigenin, various mutants of Zp were generated within
the ZIIIA, ZIIIB, Z1D and ZII domains for luciferase
analysis. TW01 cells were transfected with vector
control (pGL2) wild-type Zp (Zp-221) and six mutants
(mZIIIA, mZIIIB, mZ1D-1, mZ1D-2, ZII-1 and ZII-2).
As shown in Figure 6b, the inhibition of Zp activity
caused by apigenin was compromised significantly for
mZIIIA, mZIIIB, mZ1D-1, mZII-1 and mZII-2, while
activity of mZ1D-2 maintained the similar level as the
wild-type control (upper and lower panels).
In summary, the ZIIIA/B, Z1D and ZII elements of Zp
are important for Zp inhibition by apigenin, suggesting
that the corresponding transcription factors may
participate in Zp inhibition by apigenin.
EBV infection is prevalent worldwide and has been
strongly associated with several human malignancies.
Exploring new drugs with greater efficacy and less
cytotoxicity is an important approach to conquering this
threat. For this purpose, apigenin was identified by
screening and we found it to have an effective inhibitory
effect against EBV reactivation. Apigenin exerted great
inhibition of EBV lytic protein expression, not only in
epithelial cells but also in B cells (Figures 1 and 2). It
also repressed the numbers of EBV-reactivating cells
(Figure 3) and inhibited EBV production (Figure 4).
Further study indicated that apigenin repressed the
promoter activities of two IE genes, Zp and Rp, following
chemical and Zta/Rta induction (Figures 5). To
determine which elements of Zp were important for
inhibition, we demonstrated that the ZIIIA/B, Z1D and
ZII domains were involved in Zp inhibition by apigenin.
These results demonstrate apigenin can inhibit EBV
reactivation by repressing the promoter activities of two
Anti-EBV compounds can be divided into two major
categories: (1) those that interfere with virus-encoded
enzymes important for virus production, e.g. ACV, GCV
and BAY 57–1293 , and (2) those that interfere with
the cellular processes required for virus production, e.g.
the CDK inhibitor roscovitine . The compounds
belonging to the former category target selectively a
specific enzyme and several disadvantages have emerged,
such as viral resistance and a narrow spectrum. To avoid
this limitation, compounds targeting cellular signaling
pathways were developed as new anti-EBV drugs. As a
large family of natural compounds, the flavonoids have
been less well studied for their anti-virus functions.
Among them, apigenin was reported to inhibit
enterovirus-71 infection by disrupting viral RNA
associated factors  and it also inhibits hepatitis C virus
replication by decreasing microRNA122  and
restricts FMDV infection by inhibiting viral translational
activity . Moreover, although several flavonoids have
been reported to have anti-EBV activity, these studies
did not explore their inhibitory mechanisms and most
such studies were made using the B cell system [42, 43].
Compared with conventional agents, as an anti-viral
agent, apigenin has disadvantages such as lower
specificity; however, it has many other attractive benefits, such
as low cost, availability, safety and convenience. In
addition to apigenin, curcumin has been shown to have
an inhibitory effect on Zp to block the EBV lytic cycle in
B cells . Using a reporter assay, retinoic acid was also
found to have potent anti-EBV activity . Compared
to some other natural compounds affecting only Zp 
or Rp , apigenin is able to interfere with both Zp
and Rp activities, suggesting it may have a broader
spectrum for application.
Because Zp is the first promoter to be activated during
the EBV lytic cycle, how apigenin inhibits Zp is a key
issue for further study and application. There are eight
domains located within Zp, and various cellular factors
are involved in these domains. Among them, ZIIIA/B,
Z1D, and ZII may play regulatory roles in apigenin
inhibition (Figure 6). ZIIIA and ZIIIB have several
Zta-binding motifs in these regions, and are responsible for
TPA-induced Zp activation . For Z1D, binding
domains of Sp1/Sp3 and MEF2D have been identified in
this region and their binding has also been shown to
play important roles in EBV induction [30, 45–47]. For
the ZII region, it was found that cellular proteins
ATF1/2 are predominant factors for Zp activation in this
region, and the CREB/AP-1 family proteins are involved
in it as well . Based on our finding, Zp inhibition by
apigenin may be through interacting with Sp1/3, ATF-1/
2 and CREB (Figure 6). After reviewing the literature,
flavonoids have been found to have abilities to suppress
Sp1 activity , interact with ATF-1/2 [49–51] and
change the phosphorylation status of CREB . From
our results, we can postulate reasonably that apigenin
inhibits Zp induction likely through a complex process,
which may involve a combination of the Sp1/Sp3,
ATF1/2 and CREB pathways.
Another two possible mechanisms of apigenin
inhibition are blockage of the src, MAPK/p38 kinase pathway
and ROS generation. Cellular signaling pathways such as
protein kinase C, src or p38 kinase are necessary for
EBV reactivation [53, 54]. Recently, we found that ROS
plays a crucial role in EBV reactivation following
Nmethyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment
. Apigenin can block the src, PKC and p38 signaling
pathways [56, 57]. It is also shown to be a strong ROS
scavenger . It is reasonable to propose that apigenin
acts to inhibit EBV reactivation through some of these
mechanisms. Further studies are in progress.
Development of cancer treatments to improve survival
and the quality of life of cancer patients is an important
issue, especially during the past three decades. The
major treatments for cancer include chemotherapy,
surgery, radiation and immunotherapy. Recently, the
concept, so-called “antimicrobial adjuvant therapy”, has
been proposed to treat virus-related malignancies. For
treatment of EBV-associated cancer, induction-lytic or
anti-EBV strategies are studied for the treatment of
EBV-related malignancies. The former strategy is more
effective on regression of tumors, however, the escaping
tumor cells may become more malignant because the
expression of EBV lytic proteins in an abortive lytic state
can cause genome instability and then increased
tumorigenesis. In addition, although an anti-EBV strategy is
weak on tumor remission, the patients have less risk
from escaping EBV-lytic cells. In other words, the
antiEBV strategy is more suitable for prevention. In recent
years, the concept of chemoprevention has been growing
rapidly in oncology. This focuses on the prevention of
cancer using natural or synthetic compounds. Apigenin
is a natural plant flavone and it was first shown to have
chemopreventive properties by Birt et al. . Until
now, apigenin has been found to have several
anti-cancer functions: anti-oxidant, anti-mutagenic,
antiproliferative, anti-carcinogenic and anti-progression
properties . We believe that through these
anticancer properties, combined with the anti-EBV effect,
apigenin will provide a more profound benefit in
chemoprevention and therapy of EBV-related malignancies.
Compared to the numerous studies focused on
anticancer and antioxidation, the antiviral activities of
apigenin have been less well studied. In fact, there are
some studies suggesting that apigenin has anti-viral
activity against other viruses [40, 60]. It is worthy of
further study further to determine whether apigenin has
an inhibitory effect on various other families of viruses.
In this study, we found that the flavonoid apigenin inhibits
EBV reactivation by repressing EBV IE promoter Zp and Rp
activities. This finding may provide useful information for
drug development and apigenin may be an alternative choice
for therapy and prevention of EBV-related malignancies.
Additional file 1: The cytotoxicity of apigenin plus TPA + SB to
epithelial cells and B cells (a) NA, (b) HA and (c) P3HR1 cell lines were
pre-treated with apigenin for 1 h and then TPA + SB were added for
24 h. Cell viability was determined by WST-1 assay, as described in
Methods. The values are means ± SD from at least two separate
experiments. CC50 values also were calculated and are given at the top of
each group. (TIF 146 kb)
Additional file 2: Apigenin represses Zp activity upon HDACi induction
Control plasmid PGL2 or Zp was transfected into NA cells. Three hours
after of transfection, apigenin was added or not for pre-treatment for 1 h
and then various HDAC inhibitors, including (a) SB (3 mM); (b) TSA (5 μM)
; (c) SAHA (10 μM)  and (d) romidepsin (10 nM) , were used to
induce EBV into the lytic cycle. After induction for a further 24 h, lysates
were collected for measurement of luciferase activity. The mean and
standard deviation of each sample were calculated in duplicate from at
least two independent experiments. (TIF 100 kb)
ACV: Acyclovir; BL: Burkitt’s lymphoma; CC50: Cytotoxicity concentration;
GCV: Ganciclovir; IE proteins: Immediate early protein; MNNG:
N-methyl-N’-nitro-N-nitrosoguanidine; NPC: Nasopharyngeal carcinoma;
SB: Sodium butyrate; TPA: 12-o-tetradecanoyl-phorbol-1,3-acetate
This work was supported partly by National Health Research Institutes and
Ministry of Science and Technology, Taiwan (NSC99-3112-B-400-009,
NSC101-2325-B-400-023, NSC102-2325-B-400-021, NSC103-2325-B-400-008,
MOST104-2320-B-400 -016, MOST105-2325-B-400 -016).
Availability of data and materials
Yes, all data are fully available without restriction.
Conceived and designed the experiments: CCW JYC. Performed the
experiments: CCW HYH YJC. Analyzed the data: CCW. Gave critical
suggestion for solving experimental problems: CYF YJC SPC Contributed
reagents/materials/analysis tools: CYF SPC. Wrote the paper: CCW.
CCW and SYH are post-doctoral researchers in National Health Research
Institutes. CYF is a research fellow in Wan Fang Hospital. HYH, YJC and SPC
are research assistants in National Health Research Institutes. CHT is a
professor in National Taiwan University. JYC is an emeritus investigator in
National Health Research Institutes.
1. Rickinson AB , Kieff E. Epstein-Barr virus. In: Knipe DM , Howley PM , editors. Field's Virology. Philadelphia: Lippincott Williams & Wilkins; 2001 . p. 2575 - 627 .
2. Henle W , Henle G. Evidence for an etiologic relation of the Epstein-Barr virus to human malignancies . The Laryngoscope . 1977 ; 87 : 467 - 73 .
3. Ling W , Cao SM , Huang QH , Li YH , Deng MQ . Prognostic implication of pretreatment titer of serum immunoglobulin A against Epstein-Barr virus capsid antigen in nasopharyngeal carcinoma patients in Sihui, Guangdong . Ai Zheng . 2009 ; 28 : 57 - 9 .
4. Asito AS , Piriou E , Odada PS , Fiore N , Middeldorp JM , Long C , Dutta S , Lanar DE , Jura WG , Ouma C , Otieno JA , Moormann AM , Rochford R. Elevated antiZta IgG levels and EBV viral load are associated with site of tumor presentation in endemic Burkitt's lymphoma patients: a case control study . Infect Agent Cancer . 2010 ; 5 : 13 .
5. Chen JY , Hwang LY , Beasley RP , Chien CS , Yang CS. Antibody response to Epstein-Barr-virus-specific DNase in 13 patients with nasopharyngeal carcinoma in Taiwan: a retrospective study . Journal of medical virology . 1985 ; 16 : 99 - 105 .
6. Chien YC , Chen JY , Liu MY , Yang HI , Hsu MM , Chen CJ , Yang CS. Serologic markers of Epstein-Barr virus infection and nasopharyngeal carcinoma in Taiwanese men . N Engl J Med . 2001 ; 345 : 1877 - 82 .
7. Zeng Y , Zhang LG , Wu YC , Huang YS , Huang NQ , Li JY , Wang YB , Jiang MK , Fang Z , Meng NN . Prospective studies on nasopharyngeal carcinoma in Epstein-Barr virus IgA/VCA antibody-positive persons in Wuzhou City , China. International journal of cancer . 1985 ; 36 : 545 - 7 .
8. Cabras G , Decaussin G , Zeng Y , Djennaoui D , Melouli H , Broully P , Bouguermouh AM , Ooka T. Epstein - Barr virus encoded BALF1 gene is transcribed in Burkitt's lymphoma cell lines and in nasopharyngeal carcinoma's biopsies . J Clin Virol . 2005 ; 34 : 26 - 34 .
9. Luka J , Deeb ZE , Hartmann DP , Jenson B , Pearson GR . Detection of antigens associated with Epstein-Barr virus replication in extracts from biopsy specimens of nasopharyngeal carcinomas . Journal of the National Cancer Institute . 1988 ; 80 : 1164 - 7 .
10. Zhang JX , Chen HL , Zong YS , Chan KH , Nicholls J , Middeldorp JM , Sham JS , Griffin BE , Ng MH . Epstein-Barr virus expression within keratinizing nasopharyngeal carcinoma . Journal of medical virology . 1998 ; 55 : 227 - 33 .
11. Fang CY , Lee CH , Wu CC , Chang YT , Yu SL , Chou SP , Huang PT , Chen CL , Hou JW , Chang Y , Tsai CH , Takada K , Chen JY . Recurrent chemical reactivations of EBV promotes genome instability and enhances tumor progression of nasopharyngeal carcinoma cells . International journal of cancer . 2009 ; 124 : 2016 - 25 .
12. Fang CY , Huang SY , Wu CC , Hsu HY , Chou SP , Tsai CH , Chang Y , Takada K , Chen JY . The synergistic effect of chemical carcinogens enhances EpsteinBarr virus reactivation and tumor progression of nasopharyngeal carcinoma cells . PloS one . 2012 ; 7 : e44810 .
13. Wu CC , Liu MT , Chang YT , Fang CY , Chou SP , Liao HW , Kuo KL , Hsu SL , Chen YR , Wang PW , Chen YL , Chuang HY , Lee CH , Chen M , Wayne Chang WS , Chen JY . Epstein-Barr virus DNase (BGLF5) induces genomic instability in human epithelial cells . Nucleic acids research. 2010; 38 : 1932 - 49 .
14. Chang YH , Lee CP , Su MT , Wang JT , Chen JY , Lin SF , Tsai CH , Hsieh MJ , Takada K , Chen MR . Epstein-Barr virus BGLF4 kinase retards cellular S-phase progression and induces chromosomal abnormality . PloS one . 2012 ; 7 : e39217 .
15. Chiu SH , Wu CC , Fang CY , Yu SL , Hsu HY , Chow YH , Chen JY . Epstein-Barr virus BALF3 mediates genomic instability and progressive malignancy in nasopharyngeal carcinoma . Oncotarget . 2014 ; 5 : 8583 - 601 .
16. Hong GK , Gulley ML , Feng WH , Delecluse HJ , Holley-Guthrie E , Kenney SC. Epstein-Barr virus lytic infection contributes to lymphoproliferative disease in a SCID mouse model . Journal of virology . 2005 ; 79 : 13993 - 4003 .
17. Hong GK , Kumar P , Wang L , Damania B , Gulley ML , Delecluse HJ , Polverini PJ , Kenney SC . Epstein-Barr virus lytic infection is required for efficient production of the angiogenesis factor vascular endothelial growth factor in lymphoblastoid cell lines . Journal of virology . 2005 ; 79 : 13984 - 92 .
18. Lin JC , Nelson DJ , Lambe CU , Choi EI . Metabolic activation of 9 ([2-hydroxy1-(hydroxymethyl) ethoxy] methyl) guanine in human lymphoblastoid cell lines infected with Epstein-Barr virus . Journal of virology . 1986 ; 60 : 569 - 73 .
19. Wang FZ , Roy D , Gershburg E , Whitehurst CB , Dittmer DP , Pagano JS . Maribavir inhibits Epstein-Barr virus transcription in addition to viral DNA replication . Journal of virology . 2009 ; 83 : 12108 - 17 .
20. Sista ND , Pagano JS , Liao W , Kenney S. Retinoic acid is a negative regulator of the Epstein-Barr virus protein (BZLF1) that mediates disruption of latent infection . Proceedings of the National Academy of Sciences of the United States of America . 1993 ; 90 : 3894 - 8 .
21. Chang LK , Wei TT , Chiu YF , Tung CP , Chuang JY , Hung SK , Li C , Liu ST . Inhibition of Epstein-Barr virus lytic cycle by (−)-epigallocatechin gallate . Biochemical and biophysical research communications. 2003; 301 : 1062 - 8 .
22. Hergenhahn M , Soto U , Weninger A , Polack A , Hsu CH , Cheng AL , Rosl F. The chemopreventive compound curcumin is an efficient inhibitor of Epstein-Barr virus BZLF1 transcription in Raji DR-LUC cells . Molecular carcinogenesis . 2002 ; 33 : 137 - 45 .
23. Wu CC , Chuang HY , Lin CY , Chen YJ , Tsai WH , Fang CY , Huang SY , Chuang FY , Lin SF , Chang Y , Chen JY . Inhibition of Epstein-Barr virus reactivation in nasopharyngeal carcinoma cells by dietary sulforaphane . Molecular carcinogenesis . 2013 ; 52 : 946 - 58 .
24. Patel D , Shukla S , Gupta S. Apigenin and cancer chemoprevention: progress, potential and promise (review) . International journal of oncology . 2007 ; 30 : 233 - 45 .
25. Tsai CH , Williams MV , Glaser R. Characterization of two monoclonal antibodies to Epstein-Barr virus diffuse early antigen which react to two different epitopes and have different biological function . Journal of virological methods . 1991 ; 33 : 47 - 52 .
26. Tsai CH , Liu MT , Chen MR , Lu J , Yang HL , Chen JY , Yang CS. Characterization of monoclonal antibodies to the Zta and DNase proteins of Epstein-Barr virus . Journal of biomedical science . 1997 ; 4 : 69 - 77 .
27. Chang Y , Tung CH , Huang YT , Lu J , Chen JY , Tsai CH . Requirement for cellto-cell contact in Epstein-Barr virus infection of nasopharyngeal carcinoma cells and keratinocytes . Journal of virology . 1999 ; 73 : 8857 - 66 .
28. Hinuma Y , Konn M , Yamaguchi J , Grace Jr JT. Replication of herpes-type virus in a Burkitt lymphoma cell line . Journal of virology . 1967 ; 1 : 1203 - 6 .
29. Chen YJ , Tsai WH , Chen YL , Ko YC , Chou SP , Chen JY , Lin SF . Epstein-Barr virus (EBV) Rta-mediated EBV and Kaposi's sarcoma-associated herpesvirus lytic reactivations in 293 cells . PloS one . 2011 ; 6 : e17809 .
30. Tsai PF , Lin SJ , Weng PL , Tsai SC , Lin JH , Chou YC , Tsai CH . Interplay between PKCdelta and Sp1 on histone deacetylase inhibitor-mediated Epstein-Barr virus reactivation . Journal of virology . 2011 ; 85 : 2373 - 85 .
31. Flemington E , Speck SH. Autoregulation of Epstein-Barr virus putative lytic switch gene BZLF1 . Journal of virology . 1990 ; 64 : 1227 - 32 .
32. Liu P , Liu S , Speck SH . Identification of a negative cis element within the ZII domain of the Epstein-Barr virus lytic switch BZLF1 gene promoter . Journal of virology . 1998 ; 72 : 8230 - 9 .
33. Chiang LC , Ng LT , Lin IC , Kuo PL , Lin CC . Anti-proliferative effect of apigenin and its apoptotic induction in human Hep G2 cells. Cancer letters . 2006 ; 237 : 207 - 14 .
34. Lieberman PM , Hardwick JM , Sample J , Hayward GS , Hayward SD . The Zta transactivator involved in induction of lytic cycle gene expression in Epstein-Barr virus-infected lymphocytes binds to both AP-1 and ZRE sites in target promoter and enhancer regions . Journal of virology . 1990 ; 64 : 1143 - 55 .
35. Hui KF , Ho DN , Tsang CM , Middeldorp JM , Tsao GS , Chiang AK . Activation of lytic cycle of Epstein-Barr virus by suberoylanilide hydroxamic acid leads to apoptosis and tumor growth suppression of nasopharyngeal carcinoma . International journal of cancer . 2012 ; 131 : 1930 - 40 .
36. Hui KF , Cheung AK , Choi CK , Yeung PL , Middeldorp JM , Lung ML , Tsao SW , Chiang AK . Inhibition of class I histone deacetylases by romidepsin potently induces Epstein-Barr virus lytic cycle and mediates enhanced cell death with ganciclovir . International journal of cancer . 2016 ; 138 : 125 - 36 .
37. Kleymann G , Fischer R , Betz UA , Hendrix M , Bender W , Schneider U , Handke G , Eckenberg P , Hewlett G , Pevzner V , Baumeister J , Weber O , Henninger K , Keldenich J , Jensen A , Kolb J , Bach U , Popp A , Maben J , Frappa I , Haebich D , Lockhoff O , Rubsamen-Waigmann H. New helicase-primase inhibitors as drug candidates for the treatment of herpes simplex disease . Nature medicine . 2002 ; 8 : 392 - 8 .
38. Kudoh A , Daikoku T , Sugaya Y , Isomura H , Fujita M , Kiyono T , Nishiyama Y , Tsurumi T. Inhibition of S-phase cyclin-dependent kinase activity blocks expression of Epstein-Barr virus immediate-early and early genes, preventing viral lytic replication . Journal of virology . 2004 ; 78 : 104 - 15 .
39. Zhang W , Qiao H , Lv Y , Wang J , Chen X , Hou Y , Tan R , Li E. Apigenin inhibits enterovirus-71 infection by disrupting viral RNA association with trans-acting factors . PloS one . 2014 ; 9 : e110429 .
40. Shibata C , Ohno M , Otsuka M , Kishikawa T , Goto K , Muroyama R , Kato N , Yoshikawa T , Takata A , Koike K. The flavonoid apigenin inhibits hepatitis C virus replication by decreasing mature microRNA122 levels . Virology . 2014 ; 462 - 463 : 42 - 8 .
41. Qian S , Fan W , Qian P , Zhang D , Wei Y , Chen H , Li X. Apigenin restricts FMDV infection and inhibits viral IRES driven translational activity . Viruses . 2015 ; 7 : 1613 - 26 .
42. Iwase Y , Takemura Y , Ju-ichi M , Ito C , Furukawa H , Kawaii S , Yano M , Mou XY , Takayasu J , Tokuda H , Nishino H. Inhibitory effect of flavonoids from citrus plants on Epstein-Barr virus activation and two-stage carcinogenesis of skin tumors . Cancer letters . 2000 ; 154 : 101 - 5 .
43. Iwase Y , Takemura Y , Ju-ichi M , Mukainaka T , Ichiishi E , Ito C , Furukawa H , Yano M , Tokuda H , Nishino H. Inhibitory effect of flavonoid derivatives on Epstein-Barr virus activation and two-stage carcinogenesis of skin tumors . Cancer letters . 2001 ; 173 : 105 - 9 .
44. Chang FR , Hsieh YC , Chang YF , Lee KH , Wu YC , Chang LK . Inhibition of the Epstein-Barr virus lytic cycle by moronic acid . Antiviral research . 2010 ; 85 : 490 - 5 .
45. Chen C , Li D , Guo N. Regulation of cellular and viral protein expression by the Epstein-Barr virus transcriptional regulator Zta: implications for therapy of EBV associated tumors . Cancer biology & therapy . 2009 ; 8 : 987 - 95 .
46. Chua HH , Chiu HY , Lin SJ , Weng PL , Lin JH , Wu SW , Tsai SC , Tsai CH . p53 and Sp1 cooperate to regulate the expression of Epstein-Barr viral Zta protein . Journal of medical virology . 2012 ; 84 : 1279 - 88 .
47. Liu S , Liu P , Borras A , Chatila T , Speck SH . Cyclosporin A-sensitive induction of the Epstein-Barr virus lytic switch is mediated via a novel pathway involving a MEF2 family member . EMBO J . 1997 ; 16 : 143 - 53 .
48. Yuan H , Gong A , Young CY . Involvement of transcription factor Sp1 in quercetin-mediated inhibitory effect on the androgen receptor in human prostate cancer cells . Carcinogenesis . 2005 ; 26 : 793 - 801 .
49. Guo Z , Du X , Iacovitti L. Regulation of tyrosine hydroxylase gene expression during transdifferentiation of striatal neurons: changes in transcription factors binding the AP-1 site . J Neurosci . 1998 ; 18 : 8163 - 74 .
50. Kole L , Giri B , Manna SK , Pal B , Ghosh S. Biochanin-A, an isoflavon, showed anti-proliferative and anti-inflammatory activities through the inhibition of iNOS expression, p38-MAPK and ATF-2 phosphorylation and blocking NFkappaB nuclear translocation . Eur J Pharmacol . 2011 ; 653 : 8 - 15 .
51. Oleaga C , Ciudad CJ , Noe V , Izquierdo-Pulido M. Coffee polyphenols change the expression of STAT5B and ATF-2 modifying cyclin D1 levels in cancer cells . Oxid Med Cell Longev . 2012 ; 2012 : 390385 .
52. Yang MH , Kim J , Khan IA , Walker LA , Khan SI . Nonsteroidal anti-inflammatory drug activated gene-1 (NAG-1) modulators from natural products as anticancer agents . Life sciences . 2014 ; 100 : 75 - 84 .
53. Yu X , McCarthy PJ , Lim HJ , Iempridee T , Kraus RJ , Gorlen DA , Mertz JE . The ZIIR element of the Epstein-Barr virus BZLF1 promoter plays a central role in establishment and maintenance of viral latency . Journal of virology . 2011 ; 85 : 5081 - 90 .
54. Miller CL , Lee JH , Kieff E , Longnecker R. An integral membrane protein (LMP2) blocks reactivation of Epstein-Barr virus from latency following surface immunoglobulin crosslinking . Proceedings of the National Academy of Sciences of the United States of America . 1994 ; 91 : 772 - 6 .
55. Huang SY , Fang CY , Tsai CH , Chang Y , Takada K , Hsu TY , Chen JY . N-methylN'-nitro-N-nitrosoguanidine induces and cooperates with 12-Otetradecanoylphorbol-1,3-acetate/sodium butyrate to enhance Epstein-Barr virus reactivation and genome instability in nasopharyngeal carcinoma cells . Chemico-biological interactions . 2014 ; 188 : 623 - 34 .
56. Byun S , Park J , Lee E , Lim S , Yu JG , Lee SJ , Chen H , Dong Z , Lee KW , Lee HJ . Src kinase is a direct target of apigenin against UVB-induced skin inflammation . Carcinogenesis . 2013 ; 34 : 397 - 405 .
57. Lin JK , Chen YC , Huang YT , Lin-Shiau SY. Suppression of protein kinase C and nuclear oncogene expression as possible molecular mechanisms of cancer chemoprevention by apigenin and curcumin . Journal of cellular biochemistry . 1997 ; 28 - 29 : 39 - 48 .
58. Lin CM , Chen CT , Lee HH , Lin JK . Prevention of cellular ROS damage by isovitexin and related flavonoids . Planta medica . 2002 ; 68 : 365 - 7 .
59. Birt DF , Walker B , Tibbels MG , Bresnick E. Anti-mutagenesis and antipromotion by apigenin, robinetin and indole-3-carbinol . Carcinogenesis . 1986 ; 7 : 959 - 63 .
60. Critchfield JW , Butera ST , Folks TM . Inhibition of HIV activation in latently infected cells by flavonoid compounds . AIDS research and human retroviruses . 1996 ; 12 : 39 - 46 .