Download Effect of Pinus massoniana Lamb. bark extract on lytic cycle of

Indian Journal of Experimental Biology
Vol. 50, October 2012, pp. 708-713
Effect of Pinus massoniana Lamb. bark extract on lytic cycle of Epstein-Barr virus
Shuxia Xu1, Shimin Zhang1*, Xuedong Wang2, Yuqian Gao1, Xing Qin1 & Kun Wu1*
1
2
College of Life Science, Henan Agricultural University, Zhengzhou, Henan, China
School of Environmental Science and Public Health, Wenzhou Medical College, Wenzhou, China
Received 16 March 2012; revised 30 July 2012
Pinus massoniana bark extract (PMBE) at a concentration of 60 μg/mL or more inhibits the expression of Epstein–Barr
virus (EBV) lytic proteins, such as Rta, Zta, and EA-D. EBV lytic cycle was blocked by inhibiting the transcription of
immediate-early genes. The results suggest that the PMBE has anti-EBV activity. Thus, the extract is potentially useful in
preventing the lytic development of EBV in vitro.
Keywords: Bark extract, Epstein-Barrr virus, EA-D, Lytic cycle, Pinus massoniana, Rta, Zta
Pinus massoniana belongs to Pinaceae family and
distribution area ranges from the south regions of
China, north regions of Vietnam to India1. Its needle,
bark and turpentine have been used in Chinese folk
medicine2. Traditional Chinese medicine use bark of
P. massoniana to promote constringency, haemostasis
and detoxification, and it has been variously
prescribed for the treatment of rheumatism arthralgia,
hypertension and chilblain in China and other
countries of the Orient3. In eastern Asia (mainly
China and Japan), people made use of P. massoniana
needles and bark in diet therapies for thousands of
years4. In the last decade of the 20th century, bioactive
substances of P. massoniana bark have not been
successfully extracted through crude sieving,
centrifugation, or micro- and ultra-filtration with
hollow fiber and coiled-spiral nanofiltration
membranes4. It has been confirmed that the bioactive
substances in P. massoniana bark extract (PMBE) are
flavonoids (26.0-28.3%) (mainly procyanidins). The
active constituent of these are mainly included in the
procyanidin B series5.
In recent years, the inhibitory activity of some
flavonoids against human immunodeficiency virus
(HIV) has stimulated interest of the researchers.
Baicalin inhibits HIV-1 infection and replication in
vitro6-8. The relationship between flavonoid structure
and their inhibitory activity against HIV-1 have been
reported8,9. Flavonoids also inhibit other viruses. For
___________
Correspondent authors
Telephone: 86 371 63555790
E-mail: [email protected] (SZ); [email protected] (KW)
example, quercetin, morin, rutin, dihydroquercetin,
dihydrofisetin, pelargonidin chloride and catechin
possess activity against seven viruses, including
herpes simplex virus (HSV), respiratory syncytial
virus, poliovirus and Sindbis virus10. Antiviral effect
includes the inhibition of viral polymerase and
binding of viral nucleic acid or capsid proteins.
The Epstein Barr virus (EBV) was first described
in 1964 by Epstein, Achong, and Barr in
lymphoblastoid cell lines derived from a Burkitt’s
lymphoma tumor biopsy. EBV is a gamma herpes
virus (type 4). It is a DNA virus which infects >90%
of individuals11. The primary exposure to EBV
usually occurs in childhood. Lymphoid and epithelial
cells are infected by EBV. After infecting
B lymphocytes, EBV is typically latent. This virus
causes infectious mononucleosis and is very relevant
with several malignant diseases, including Burkitt’s
lymphoma, T-cell lymphoma, Hodgkin’s disease,
gastric cancer and nasopharyngeal carcinoma11.
Among these diseases, infectious mononucleosis is an
important disease and closely related to the EBV lytic
activation and spread of the virus in the body12.
However, the virus has to enter a lytic cycle to
proliferate. This investigation demonstrates the
inhibitory effect of PMBE is not only on EA-D protein
expression but also on the immediate-early proteins of
EBV, thus, blocking the EBV lytic cascade.
Materials and Methods
An EBV-positive Burkitt’s lymphoma cell line,
P3HR1, was cultured in RPMI 1640 medium
supplemented with 10% fetal calf serum. EBV lytic
XU et al.: PINUS MASSONIANA BARK EXTRACT & EPSTEIN-BARR VIRUS
cycle was induced with 300 nM trichostatin A (TSA)
(Sigma, China).
Preparation of P. massoniana bark extracts
(PMBE)Bark from P. massoniana collected from
Wuhan, Hubei Province, China, was dried, ground into
powder, and put into a diffuser. Distilled water was
added to the diffuser, which was then heated for filter.
The filters were subjected to crude sieving,
centrifugation and microfiltration for removal of
suspended matter and macromolecular impurities.
Hollow fibre membrane ultrafiltration was used to
generate permeation solution containing bioactive
compounds such as flavonoids and proanthocyanidins.
This solution was inspissated with a coiled-spiral
nanofiltration membrane for removal of micromolecule
impurities such as carbohydrates, inorganic solutes and
most of the water. The resulting concentrate was dried
to yield biologically active PMBE. For production of a
stock solution, 100 mg PMBE was dissolved in 10 mL
absolute ethanol or DMSO, filtered through a 0.22 µm
Millipore membrane and stored in 5 mL Eppendorf
tubes at 4 °C. For production of a working solution, the
stock solution was diluted in RPMI 1640 culture
medium containing 10% FBS, 100 units/mL penicillin,
100 µg/mL streptomycin and 10 mM HEPES. Working
solutions of 10, 20, 40, 60, 80 µg PMBE/mL were
made for MTT proliferation assays in human liver
cancer and normal cell lines.
High-performance liquid chromatography (HPLC)
analysisAn Alliance HPLC system (HP1100, USA)
consisting of an integrated controller, pump, auto
injector, and a photodiode array detector were used. A
prepacked Diamonsil TMC18 HPLC column (250×4.6
mm, 5 m particle size) with a flow rate of 0.8 mL/min,
and a mobile phase consisting of methanol (A) and
water (B), were continuously degassed. A gradient
elution was performed using the following solvent
gradient, from 20A/80B to 80A/20B in 20 min. UV
detection was performed at 280 nm, and all the samples
were injected in triplicate. Chromatographic data were
processed and the taxifolin content was calculated in
PMBE.
Cell line and induction of EBV lytic cycleAn
EBV-positive Burkitt’s lymphoma cell line, P3HR1,
was cultured in RPMI 1640 medium supplemented
with 10% fetal calf serum. EBV lytic cycle was
induced with 300 nM trichostatin A (TSA) (Sigma,
China).
Immunoblot analysisProteins resolved by
electrotransferred SDS-polyacrylamide gel to Hybond
709
C membrane (Amersham Phar-macia Biological
Technology) at 90V for 1.5 h, were detected by
respective antibody. The proteins were blotted and
detected as per the instructios of ECL test kit
(Biological Industry, Israel). Monoclonal antibodies
against Zta and Rta proteins were procured from
Argene (Varilhes, France), whereas, monoclonal
antibody against EA-D protein was purchased from
Advanced Biotechnology (Columbia, MD).
Indirect immunofluorescence analysisP3HR1
cells (2×106) were washed with PBS, followed by
fixing with 4% paraformaldehyde for 30 min, and
finally washed with PBS containing 0.1 % Triton
X-100 for 5min. Cells were then washed with PBS,
treated with 1 % BSA in PBS for 1 h, and incubated
with 1:2000-diluted anti-Rta or l anti-Zta or antiEA-D monoclonoal antibodies for 1 h at 37 °C. Then
the cells were washed with PBS, treated with 0.5 %
Tween-20 in PBS, and incubated with 1:2000-diluted
FITC-conjugated goat anti-mouse immunoglobulin G
(KPL, Guildford, UK) for 1 h at 37 °C to detect EAD, Rta, and Zta proteins. Beta-actin was used as a
control. Finally, cells were resuspended in 1%
paraformaldehyde and analyzed on a fluorometer
(Becton–Dickinson, USA).
Indirect immunofluorescence analysisP3HR1
cells were treated with antibodies as described for the
flow cytometry analysis, except that the cells were
plated on poly-L L-lysine (Sigma Chemical)-coated
cover slips and fixed with 4% paraformaldehyde.
Finally, fluorescence was observed with a Zeiss
Axioskop 20 fluorescence microscope. Images were
captured with a charge-coupled device camera and
processed by the Image-Pro Plus, version 4.5 software
(Media Cybernetics the Imaging Experts, Maryland).
Transfection and luciferase assayFor transfection
study, plasmids were prepared by CsCl gradient
centrifugation. Transfection luciferase activities were
operated according to measurement method16. For
plasmid transfection, 10 μg of plasmid DNA was
mixed with 5 × 106 cells in 300 mL of culture medium.
Electroporation was performed at 960 μF and 0.2 V
with a Bio-Rad (Richmond, Calif.) Gene-Pulser
electroporator. To closely examine the gene expression
under latent conditions, cells were transferred to 10 mL
of fresh culture medium. Cell lysate was prepared 24 h
after transfection. Briefly, cells were harvested by
centrifugation and were washed with phosphate
buffered saline. Cells were then lysed in 200 μL
of lysis buffer containing 25 mM Tris-phosphate
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INDIAN J EXP BIOL, OCTOBER 2012
(pH 7.8), 2 mM dithiothreitol, 2 mM 1,2-diaminocyclohexane- N, N, N′, N′-tetraacetic acid, 10%
glycerol, and 1% Triton X-100. The lysate was
centrifuged with a microcentrifuge at 11,750 g for
5 min at room temperature, and the supernatant was
used for the determination of luciferase activity.
Luciferase assay solution (100 μL), containing 20 mM
Tricine, 1.07 mM (MgCO3)4Mg(OH)2 .5H2O, 2.67 mM
MgSO4, 0.1 mM EDTA, 33.3 mM dithiothreitol, 270
μM coenzyme A, 470 μM luciferin, and 530 μM ATP,
was automatically injected into a test tube containing
20 μL of the cell lysate. Luciferase activity was then
monitored for 10 sec with a luminometer (Berthod, Bad
Wildbad, Germany). Each transfection experiment was
repeated at least three times, and each sample in the
experiment was prepared in duplicate.
Inhibition of the transcription of EBV immediateearly genesWith transient transfection assay, we
assessed the transcription of EBV immediate-early
genes by PMBE effect. By electroporation with 10 μg
of pRluc P3HR1, cells (5×106) were transfected, a
firefly luciferase gene (luc) transcribed from the
BRLF1 promoter was contained in the reporter
plasmid. The cells were then treated with 300 nM TSA
and 60 or 80 μg/mL PMBE immediately following
transfection. Using a method described previously,
luciferase activity expressed from the plasmid was
subsequently analyzed13. Since TSA treatment induces
the EBV lytic cycle and activates the BRLF1
promoter14, the luciferase activity exhibited by the cells
increased 3.4 times by 24 h post transfection (Fig. 4A).
On the other hand, treating the cells with PMBE
(60μg/mL）reduced the luciferase intensity to the
background level (Fig. 4A). The transcription of the
BZLF1 promoter in a transient transfection was also
inhibited by PMBE. The transcription from a luciferase
reporter plasmid, pZluc, containing a luc gene
transcribed from the BZLF1 promoter, was activated
by the TSA treatment by 8.6 times at 24 h post
transfection (Fig. 4C). Even, when the cells were
treated with 60 μg/mL PMBE, the transcription
decreased to the background level (Fig. 4C). Higher
level of PMBE at 80 μg/mL PMBE also inhibited
transcription from these two promoters (Fig. 4D). This
results showed the inhibition of transcription of BRLF1
and BZLF1 by PMBE (Fig. 4), thereby suppressing the
lytic cycle of EBV to produce EBV particles.
Results
HPLC analysis of PMGEAt least nine
components were determined in PMGE sample by
using HPLC including taxifolin, epicatechin and
epigallocatechin galloate (data not shown) (Fig. 1b).
The retention time of standard toxifolin was detected
at 5.79 min (Fig. 1a).
Inhibition of the expression of EBV lytic proteins
by PMBEPMBE inhibited the lytic cycle of EBV.
P3HR1 cells were first treated with PMBE for 1 h
prior to treatment with 300 nM TSA to activate the
EBV lytic cycle. Inhibition of the expression of EBV
lytic proteins by PMBE was assessed by immunoblot
analysis, which was performed using 1,1000-diluted
monoclonal anti-EA-D antibody. The results showed
that EA-D expression was induced by 300 nM TSA
(Fig. 2) and the expression of EA-D was completely
suppressed by PMBE at 80 μg/mL.
Indirect immunofluorescence analysis of EBV
protein expressionThe inhibitory effects of PMBE
on EBV protein expression was also analyzed by
indirect immunofluorescence analysis. Rta, Zta, and
EA-D were expressed at 24 h post TSA treatment
(Fig. 3). However, the expression of these proteins
was suppressed by treating with PMBE, both at 60
and 80 μg/mL concentration at 24 h post induction
(Fig. 3). The results showed that suppression was
dose dependent and at 80 μg/mL, there was a
complete inhibition of EBV protein expression.
Fig. 1Reverse-phase HPLC analysis of PMGE. The detection
wavelength was 280 nm. Y axis represents absorb light value, and
X value represents injection time (min).
XU et al.: PINUS MASSONIANA BARK EXTRACT & EPSTEIN-BARR VIRUS
711
Inhibition of transcription of EBV immediate-early
genesThe transcription inhibition of EBV
immediate-early genes by PMBE was assessed by
transient transfection assay. By electroporation with
10 μg of pRluc P3HR1, cells (5×106) were
transfected, a firefly luciferase gene (luc) transcribed
from the BRLF1 promoter was contained in the
reporter plasmid. The cells were then treated with
300 nM TSA and 60 or 80μg/mL PMBE immediately
following transfection. Using a method described
previously, luciferase activity expressed by the
plasmid was subsequently analyzed. Since TSA
treatment induces the EBV lytic cycle and activates
the BRLF1 promoter, the luciferase activity exhibited
by the cells increased 3.4 times after 24 h post
transfection (Fig. 4a). On the other hand, treating the
Fig. 2Immunoblot analysis of expression of Rta, Zta and EA-D
during lytic cycle by PMBE treatment. [P3HR1 cells were treated
with PMBE at different concentrations for 1 h. For inducing EBV
lytic cycle, P3HR1 cells were untreated (a) or treated (b) with 300
nM TSA. Beta-actin was treated as control].
Fig. 3Inhibitory effects of PMBE on the expression of EBV lytic
genes by indirect immunofluorescence analysis. [For inducing EBV
lytic cycle, P3HR1 cells were treated with TSA. P3HR1 cells
treated as negative control, and TSA-treated cells were as positive
control. TSA-treated cells were also pre-exposed to 60 μg(PMBE60) and 80 μg/mL (PMBE-80) PMBE. After incubation for 24 h,
cells were processed using monoclonal antibodies of anti-Rta, antiZta, and anti-EA-D for indirect immnofluorescence. In this figure,
each panel contains approximately the same number of cells].
Fig. 4Assay of activity of the BRLF1 and the BZLF1 promoter
with transient transfection. [P3HR1 cells (■) were transfected with
reporter plasmids pRluc and pZluc (10 μg). Then, cells were treated
with 300 nM TSA (▨), with 60 μg/mL: (a, c) or 80 μg/mL (b, d) of
PMBE, and finally with 300 nM of TSA (). Y axis represents
luciferase activity. The luciferase activity exhibited by the plasmids
was monitored at 24 h after treatment. Each transfection was
repeated thrice and each sample was prepared in duplicate in the
experiment].
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INDIAN J EXP BIOL, OCTOBER 2012
cells and PMBE（60 g/mL） reduced the luciferase
intensity to the background level (Fig. 4a). The
transcription of the BZLF1 promoter in a transient
transfection was also inhibited by PMBE. The
transcription from a luciferase reporter plasmid,
pZluc, containing a luc gene transcribed from the
BZLF1 promoter, was activated by the TSA treatment
by 8.6 times 24 h post transfection (Fig. 4c). Even,
when the cells were treated with 60 μg/mL PMBE, the
transcription decreased to the background level
(Fig. 4c) and 80μg/mL PMBE inhibited transcription
completely from these two promoters (Fig. 4d). These
results indicated that the transcription of BRLF1 and
BZLF1 were inhibited by PMBE (Fig. 4), thereby
suppressing the lytic cycle of EBV particles.
Discussion
Epstein-Barr virus causees diseases like infectious
mononucleosis, Burkitt’s lymphoma, Hodgkin’s
disease, nasopharyngeal carcinoma, post-transplant
lymphoproliferative disease, oral hairy leukoplakia,
and acquired immune deficiency syndrome (AIDS)
immunoblastic lymphomas11. Most of the EBVinduced pathology is attributed to viral latency11. The
latest research have implicated reactivation of lytic
virus, and specifically the lytic activator protein Zta,
in tumorigenesis and autoimmune disease15. During
primary infection, lytic replication is also required for
the foundation of latency in the host16. Hence, antiherpes drugs that are commonly used such as
acyclovir and ganciclovir, are nucleoside analogs,
suppress the function of herpes DNA polymerase and
viral lytic DNA replication.
An earlier study demonstrated that EBV virions
that are produced by B lymphocytes exhibit a tropism
by which they preferentially infect epithelial cells.
Therefore, the lytic reactivation of EBV in B
lymphocytes may be an important step towards the
oncogenesis of epithelial cells that is associated with
EBV, including nasopharyngeal carcinoma17.
Therefore, development of an effective strategy to
inhibit the lytic cycle may be of value in reducing the
risk of the disease.
There are two life cycles of EBV. After infecting B
lymphocyte cells, the virus will be maintained in
potential conditions. Before proliferation, EBV must
undergo a lytic productive cycle. In the present study,
the combination of 300 nM TSA and 80 μg/mL
PMBE was toxic to P3HR1 cells and resulted in
40–50 % cell death 24 h post treatment. In addition,
insufficiency of EA-D protein expression could be
attributed to decrease in live P3HR1 cells. Low
concentration of PMBE had less toxic effect on
P3HR1 cells. EBV immediate-early proteins like Rta
and Zta were necessary for transcription of the EA-D
gene, BMRF1. In the present study, inhibitory effect
of PMBE on Rta and Zta expression was investigated.
Results revealed that the PMBE at a concentration of
60 μg/mL inhibited the TSA-induced Rta and Zta
expression (Fig. 1), thereby, supressig the
transcription of EA-D gene, BMRF1.
The mechanism of inhibition of transcription of
BRLF1 and BZLF1 by PMBE has not been known4.
However, it is speculated that the andrographolide
inhibits the functions of the transcription factors that
activate the transcription of BRLF1 and BZLF1. In
addition, the transcription of BRLF1 and BZLF1 is
also known to be affected by the activation of various
signaling pathways, including p38 and Jun N-terminal
kinase18. However, further investigation is warranted
to determine the inhibitory effect of andrographolides
on signaling pathways that are known to activate the
transcription of EBV immediate-early genes. As of
now, influence of the pathway on the expression of
BRLF1 and BZLF1 is unknown.
Further, PMBE possesses antioxidant properties
like free radical scavenging and superoxide radical
scavenging activities4. It has been reported that the
levels of malondialdehyde in L-02 cells and aspartate
aminotransferase, alanine aminotransferase, and
alkaline phosphatase in mice were significantly
inhibited by PMBE. The main bioactive substances in
PMBE were flavonoids (mostly procyanidins).
Flavonoids have been known for their antiviral
activites. The antiviral activity of flavonoids has been
recognised since the 1940s, but in the last 2½ decades,
researchers have attempted to modify flavonoids
synthetically to improve their antiviral activities.
Accordingly several copounds have been synthesized
and one among them is 6, 4-dichloroflavan. The
possible mechanism of action of flavinoids on viruses
are either the inhibition of viral polymerase, or
binding of viral nucleic acid or viral capsid proteins.
Apart from these three proanthocyanidins from
Pavetta owariensis are inhibitory to herpes simplex
virus in vitro and coxsackie B virus. It has also been
reported that the two of the flavonoids from propolis,
chrysin and kaempferol, inhibit replication of HSV,
human coronavirus and rotavirus. Similarly, flavonol
galangin has been reported to possess significant
XU et al.: PINUS MASSONIANA BARK EXTRACT & EPSTEIN-BARR VIRUS
antiviral activity against HSV. Therefore, PMBE has
a great potential as an antiviral compound in the time
to come.
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