Betulinic acid-mediated inhibitory effect on hepatitis B
virus by suppression of manganese superoxide
dismutase expression
Dachun Yao
1,2
, Huawen Li
3
, Yulan Gou
1,4
, Haimou Zhang
5
, Athanasios G. Vlessidis
2
, Haiyan Zhou
4
,
Nicholaos P. Evmiridis
2
and Zhengxiang Liu
1
1 Internal Medicine of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
2 Laboratory of Analytical Chemistry, Department of Chemistry, University of Ioannina, Greece
3 Department of Nutrition and Food Hygiene, Guangdong Medical College, China
4 The First Hospital of Wuhan, China
5 School of Life Sciences, Hubei University, Wuhan, China
Hepatitis B virus (HBV) infection is a prevalent health
problem, affecting 350 million people worldwide; it
causes acute and chronic hepatitis, some cases of
which may progress into cirrhosis and hepatocellular
carcinoma [1]. Chronic HBV patients are currently
treated with interferon or some nucleotide analogs,

motif on the SOD2 promoter. SOD2 overexpression abolished the inhibi-
tory effect of BetA on HBV replication, whereas SOD2 knockdown mim-
icked this effect, indicating that BetA-mediated HBV clearance was due to
modulation of the mitochondrial redox balance. This observation was fur-
ther confirmed in HBV-transgenic mice, where both BetA and PC crude
extracts suppressed SOD2 expression, with enhanced reactive oxygen
species generation in liver tissues followed by substantial HBV clearance.
We conclude that BetA from PC could be a good candidate for anti-HBV
drug development.
Abbreviations
BetA, betulinic acid; CREB, cAMP-response element-binding protein; DiOC
6,
3,3¢-dihexiloxadicarbocyanine; HBeAg, hepatitis B external core
antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HBx, hepatitis B virus X protein; MMP, mitochondrial membrane
potential; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; PC, Pulsatilla chinensis; PKA, protein kinase A; PKD, protein
kinase D; ROS, reactive oxygen species; siCREB, small interfering RNA for cAMP-response element-binding protein; siRNA, small interfering
RNA; siSOD2, small interfering RNA for manganese superoxide dismutase; SOD2, manganese superoxide dismutase; TUNEL,
deoxynucleotidyl transferase dUTP nick end labeling; WT, wild-type.
FEBS Journal 276 (2009) 2599–2614 ª 2009 The Authors Journal compilation ª 2009 FEBS 2599
including some herbs, have been used for centuries to
treat viral hepatitis, but they are still not widely
accepted by conventional medicine, owing to the lack
of mechanisms and purity of herbs [2].
Pulsatilla chinensis (PC) is a traditional Chinese herb
used for the treatment of amoebic diseases, vaginal
trichomoniasis, and bacterial infections, owing to its
antiamoebic, antibacterial and antitrichomonal activi-
ties [3]. Recently, this herb was used for the treatment
of a hepatitis B patient, according to an old recipe in a
specific area of China (Yichang, Hubei), with satisfac-

[15,16], have been studied extensively for the regulation
of SOD2 expression, whereas there are few reports on
the role of cAMP-response element-binding protein
(CREB) in SOD2 expression [17,18]. CREB binds via
its basic leucine zipper domain as a dimer to cAMP
response elements containing the consensus motif
5¢-TGACGTCA-3¢; these are present in the promoters
of many genes in which transcription rates are strongly
regulated by cAMP. CREB stimulates cellular gene
transcription via the protein kinase A (PKA)-mediated
phosphorylation of CREB at Ser133 [19]. Ser133 phos-
phorylation of CREB, in turn, promotes recruitment
of the coactivator paralogs CREB-binding protein and
p300 via a kinase-inducible domain in CREB, which
appears to be sufficient for the induction of cellular
genes [20,21]. On the other hand, inhibition of CREB
phosphorylation or dephosphorylated CREB may be a
negative regulator of CREB-responsive genes [22,23].
In an effort to investigate the mechanism of the
inhibitory effect of BetA on HBV, BetA was isolated
from PC to treat hepatocytes from HBV-transgenic
mice. We found that SOD2 was downregulated by
BetA-induced CREB dephosphorylation at Ser133
through the CREB-binding motif on the SOD2 pro-
moter. SOD2 suppression-mediated ROS generation
subsequently inhibited HBV replication, decreased
HBV X protein (HBx) total level, and translocated
HBx to the mitochondria followed by cytochrome c
release. Overexpression of SOD2 totally abolished the
BetA-mediated HBV-inhibitory effect, whereas SOD2

)1
), the
DNA synthesis rate of HBV-infected cells was sub-
stantially decreased, but WT cells showed no signifi-
cant decrease. On the other hand, when the BetA dose
was even higher (20 lgÆmL
)1
), the DNA synthesis rate
of HBV-infected cells was substantially inhibited,
whereas no difference was found in WT cells, indi-
cating that BetA-induced cytotoxicity was specific
to HBV-infected cells.
Betulinic acid inhibits hepatitis B virus D. Yao et al.
2600 FEBS Journal 276 (2009) 2599–2614 ª 2009 The Authors Journal compilation ª 2009 FEBS
96 84
72
60
48
36 24 12 0
2
3
3.5
A C
B D
E F
G H
2.5
1.5
2
3

100
0
300
200
100
400
300
200
100
0 µg·mL
–1
10 µg·mL
–1
15 µg·mL
–1
20 µg·mL
–1
[3H]-thymidine incorporation
(% control)
WT
WT HBV
WT
HBV
WT
HBV
WT
HBV
WT
HBV
HBV

Caspase-3 activity
(pmol
–1
·min
–1
·mg
–1
)
*
*
0
5
20
15
10
Apoptosis rate (%)
*
*
Fig. 1. BetA-mediated selective effect on HBV-infected hepatocytes. (A) WT or HBV-infected (HBV) hepatocytes were treated with different
doses of BetA for 48 h, and cell viability was measured. (B) Cells were treated with 15 lgÆmL
)1
BetA for different times, and cell viability
was measured. (C) Cells were treated with different doses of BetA as indicated for 48 h, and then incubated with [
3
H]thymidine for 2 h to
measure the inhibitory effect of BetA on cell differentiation by the [
3
H]thymidine incorporation assay. *P < 0.05 versus WT; –P < 0.05
versus 0 lgÆmL
)1

and caspase-3 activity (Fig. 1H) were assessed. The
results showed that BetA substantially increased the
apoptosis rate and caspase-3 activity in HBV-infected
cells as compared with WT cells.
BetA-mediated selective SOD2 suppression in
HBV-infected hepatocytes
In order to clarify the effect of BetA, a microarray
assay after treatment with 15 lgÆmL
)1
BetA for 48 h
was conducted. BetA specifically decreased SOD2
mRNA expression in HBV-infected cells, whereas little
difference was seen in WT cells (data not shown).
Real-time PCR was performed for confirmatory pur-
poses, and suggested that the SOD2 mRNA level was
decreased about  2.4-fold in HBV-infected cells trea-
ted with BetA as compared with the control, but
showed no difference in WT cells (Fig. 2A). Western
blotting to measure the protein level (Fig. 2B) showed
a significant decrease in SOD2 protein in HBV-infected
cells after BetA treatment, but no change in WT cells.
SOD2 enzyme activity (Fig. 2C) decreased significantly
in HBV-infected cells after BetA treatment, whereas
little difference was found in WT cells.
The BetA-mediated SOD2 transcriptional
response element was located at the
CREB-binding site (nucleotide )1335) on the
SOD2 promoter
The mechanism of BetA-mediated SOD2 suppression
was investigated further. To localize the regulatory

0
400
300
200
100
WT HBV
WT HBV
WT HBV
SOD2 mRNA level by qPCR
(Arbitrary units)
CTL
BetA
CTL
BetA
CTL
BetA
¶
*
SOD2 protein by Western blot
(Arbitrary units)
¶
*
SOD2 protein activity
(Arbitrary units)
¶
*
Fig. 2. BetA-mediated selective SOD2 suppression in HBV-infected
hepatocytes. The 80% confluent WT or HBV-infected cells were
treated with 15 lgÆmL
)1

BetA-mediated SOD2 suppression is due to
BetA-induced CREB dephosphorylation
We have shown transcriptional activities of SOD2
that responsible to BetA treatment is due to the exis-
tence of CREB-binding elements on SOD2 promoter.
Here, we further confirmed the CREB-binding activ-
ity through chromatin immunoprecipitation analysis,
as shown in Fig. 4A. After immunoprecipitation and
reversal of the crosslinking, the endogenous SOD2
promoter was enriched by real-time PCR amplifica-
tion, using specific primers that cover the CREB-
binding motif. The results showed that the PCR
product was decreased to 47% after BetA treatment
as compared with the control group, and that the
effect was totally abolished by CREB overexpression
in the presence of BetA, whereas CREB knockdown
(siCREB) mimicked the effect. As it is well known
that CREB activity mainly depends on phosphoryla-
tion at Ser133, we next measured the levels of both
CREB protein and CREB protein phosphorylated at
Ser133 (pCREB). As shown in Fig. 4B,C, the total
CREB protein level did not change after BetA treat-
ment as compared with control, whereas the pCREB
level decreased by 42%. On the other hand, over-
expression of CREB in the presence of BetA
increased the CREB level 1.7-fold, but did not
increase the pCREB level, whereas knockdown of
CREB (siCREB) decreased the levels of both CREB
protein and pCREB. Using the above treatment, we
next measured the SOD2 mRNA level (Fig. 4D) and

(Arbitrary units)
CTL
Bet A
*
*
*
#
#
*
*
*
0
BERC
cuL
TCT
G
CAGT
T
CTGCAGT
TCTGTA
G
T
BERC
0
c
u
L
BERC
0
cuL

T
CTGCAGT
TCTGTA
G
T
BERC
c
u
L
BERC
c
u
L
BERC BERC
c
uL
c
uL
Luc
+ 308
BERC
+ 308
cuL
BERC
cuL
BERC BERC
cuL cuL
SOD2 transcriptional activity
(Arbitrary units)
*

presence of BetA, but was mimicked by siCREB.
This indicates that SOD2 expression is regulated by
CREB phosphorylation at Ser133. As we had
already shown that BetA treatment decreased CREB
phosphorylation at Ser133 (Fig. 4C), we next per-
formed in vitro experiments to determine whether
BetA could inhibit CREB phosphorylation directly.
As shown in Fig. 4F, the purified CREB was sub-
stantially phosphorylated at Ser133 in the presence
of PKA, whereas phosphorylation was markedly
inhibited by BetA, indicating that BetA could
directly inhibit CREB phosphorylation, and this
SOD2 mRNA level by qPCR
(Arbitrary units)
*
*
0
150
A
B
C
D
E
F
100
50
0
300
200
200

(Arbitrary units)
*
*
¶
CREB
PKA
BetA
+++
++–
+––
CTL BetA siCREBBetA/CREB
CTL BetA siCREBBetA/CREB
CTL BetA siCREB
BetA/CREB
CTL BetA siCREBBetA/CREB
CTL BetA siCREBBetA/CREB
Fig. 4. BetA-mediated SOD2 suppression was due to direct inhibition of CREB phosphorylation. (A) HBV-infected hepatocytes were treated
with control (CTL), 15 lgÆmL
)1
BetA, BetA with CREB overexpression (BetA ⁄ CREB›) or siRNA for CREB (siCREB) for 48 h; the chromatin
from treated cells was immunoprecipitated with CREB antibody, and the SOD2 promoter that covers the CREB-binding motif was amplified
by quantitative PCR (qPCR). (B–E) The cells treated as above were used for measurement of CREB protein level (B), pCREB protein level
(C), SOD2 mRNA level (D), and SOD2 protein level (E). *P < 0.05 versus CTL for (A)–(E). (F) In vitro-purified proteins were phosphorylated
by PKA in the presence or absence of BetA, and pCREB was measured by western blotting. *P < 0.05 versus first panel; –P < 0.05 versus
second panel.
Betulinic acid inhibits hepatitis B virus D. Yao et al.
2604 FEBS Journal 276 (2009) 2599–2614 ª 2009 The Authors Journal compilation ª 2009 FEBS
decreased amount of phosphorylated CREB (or
decreased CREB activity) downregulates SOD2
expression through the CREB-binding motif on the

tosis rate (Fig. 5D) in WT cells, and a similar effect
was observed in WT hepatocytes overexpressing
HBx; overexpression of CREB could not abolish this
effect, suggesting that the BetA-induced basal toxic
effect in WT cells is not due to BetA-induced SOD2
suppression, and HBx alone is not directly involved
in BetA-induced basal toxicity. On the other hand,
the BetA-induced toxicity was substantially increased
in HBV-infected hepatocytes as compared with WT
cells, and this effect was mostly abolished by overex-
pression of CREB, suggesting that, in HBV-infected
cells, BetA-induced toxicity is due to SOD suppres-
sion. We next measured SOD2 expression in different
0
300
A B C
D
E
F
200
100
Total lysate Mitochondria Cytosol
HBx protein level by Western blot
(Arbitrary units)
CTL
BetA
siCREB
*
*
*

BetA
*
*
*
*
*
¶
*
0
300
200
100
Total lysate Mitochondria Cytosol
CTL
BetA
siCREB
CytC protein level by Western blot
(Arbitrary units)
*
*
*
*
HBV
CTL BetA
HBx protein in mitochondria by Western blot
(Arbitrary units)
*
?
0
400

cells; as shown in Fig. 5E, the basal level of SOD2
was not changed in WT cells or WT hepatocytes
overexpressing HBx, whereas the SOD2 level was
substantially increased in HBV-infected cells as com-
pared with WT cells; this increase was totally nor-
malized by BetA, and overexpression of CREB
minimized the effect of BetA, suggesting that BetA-
induced toxicity in HBV-infected cells is due to
BetA-mediated SOD suppression. Finally, we mea-
sured HBx translocation to mitochondria in different
cells, as shown in Fig. 5F. In WT hepatocytes over-
expressing HBx, HBx was not found in mitochondria
at all in the presence of BetA (data not shown),
whereas in HBV-infected cells, BetA-induced HBx
translocation was totally abolished by CREB overex-
pression, suggesting that BetA-induced SOD2
suppression and subsequent ROS generation is the
driving force for HBx transcloation to mitochondria.
The BetA-mediated inhibitory effect on HBV is
due to SOD2 suppression and subsequent ROS
generation
We previously found that BetA suppresses SOD2
expression by inhibiting CREB phosphorylation, with
subsequent ROS overgeneration. Here, we further
investigated the potential effect of SOD2 on HBV
replication. The HBV-infected hepatocytes were trea-
ted with BetA, or BetA with SOD2 overexpression,
or siRNA for SOD2 (siSOD2) alone, and the related
biomedical parameters were measured. As shown in
Fig. 6, the levels of SOD2 mRNA (Fig. 6A) and

100
0
200
100
0
200
100
SOD2 mRNA level by qPCR
(Arbitrary units)
0
200
100
300
200
100
SOD2 mRNA level by Western blot
(Arbitrary units)
*
?
*
0
ROS formation
(Arbitrary units)
*
*
0
30
20
10
Apoptosis rate

protein level. (C) ROS generation. (D) Apopto-
sis rate determined by TUNEL assay. (E)
HBsAg secreted from cell culture medium.
(F) HBeAg secreted from cell culture med-
ium. (G) HBV DNA from treated cells was
measured by real-time quantitative PCR. (H)
HBx protein level was measured by western
blotting and quantitated. *P < 0.05 versus
the CTL group.
Betulinic acid inhibits hepatitis B virus D. Yao et al.
2606 FEBS Journal 276 (2009) 2599–2614 ª 2009 The Authors Journal compilation ª 2009 FEBS
and apoptosis (Fig. 6D); BetA treatment substantially
increased ROS generation and the apoptosis rate,
SOD2 overexpression in the presence of BetA mini-
mized the effect, and siSOD2 mimicked the effect of
BetA. We next measured the effect of these treat-
ments with different SOD2 expression levels on HBV
replication; the results showed that BetA alone sub-
stantially inhibited HBV replication, including hepati-
tis B surface antigen level (HBsAg) level (Fig. 6E),
hepatitis B external core antigen (HBeAg) level
(Fig. 6F), HBV DNA (Fig. 6G), and HBx protein
expression (Fig. 6H), whereas a combination of BetA
and SOD2 overexpression totally abolished the BetA-
mediated HBV-inhibitory effect. On the other hand,
SOD2 knockdown (siSOD2) mimicked the BetA-
induced inhibitory effect. This suggests that BetA-
induced ROS generation plays an important role in
HBV inhibition; scavenging of ROS by overexpres-
sion of the antioxidant enzyme SOD2 might not

HBV replication in vitro by SOD2 suppression, which
is similar to the effect that PC had in hepatitis B
patients in our preliminary observation (data not
shown). Here, we used HBV-transgenic mice to
determine whether BetA could achieve the same
inhibitory effect. As shown in Fig. 7A, both BetA
and PC significantly reduced HBsAg and HBeAg
serum levels and HBV DNA replication. Also, both
BetA and PC substantially decreased SOD2 mRNA
expression, whereas CREB mRNA showed no
changes (Fig. 7B). In addition, protein levels of
SOD2 and pCREB were substantially reduced after
BetA and PC treatment, whereas no changes were
found in CREB total protein level (Fig. 7C). We
also examined the enzymatic activities, and showed
that both BetA and PC not only decreased SOD2
activity, but also increased caspase-3 activity, indicat-
ing increased cytotoxicity with apoptosis rate
(Fig. 6D). Finally, we examined the levels of super-
oxide release in different tissues (Fig. 6E,F); both
BetA and PC specifically increased superoxide anion
generation in liver tissue, but had little effect in
aorta, and no effect at all in kidney and brain, indi-
cating that both BetA-mediated and PC-mediated
HBV inhibition are due to specifically decreased
SOD2 expression with subsequent ROS generation in
liver tissue.
Discussion
This study demonstrates that BetA inhibits HBV repli-
cation by suppression of SOD2 expression with subse-

induce mitochondrial dysfunction and apoptosis [24].
Given the fact that HBV-infected cells are more sus-
ceptible to BetA-induced SOD2 suppression, and
BetA-induced SOD2 suppression could directly inhibit
HBV replication, as shown in Fig. 6, we conclude that
BetA could be a good candidate for anti-HBV drug
development.
BetA-mediated CREB dephosphorylation
As BetA could cause CREB dephosphorylation at
Ser133 both in vivo and in vitro, and a mutated form
of CREB with an Ala substitution for Ser133 has been
reported to be a negative transcriptional regulator,
BetA-induced dephosphorylation of CREB could act
as a repressor of SOD2 gene transcription directly [25].
CREB, as a direct substrate of both PKA [21,26] and
protein kinase D (PKD) [27], could be phosphorylated
0
100
200
Protein level by Western blot
(Arbitrary units)
CLT BAte PC
*
*
*
*
0
50
100
150

*
0
100
200
300
400
Enzyme activity (Arbitrary units)
CTL
BetA
PC
*
*
*
*
Caspase-3 SOD2
Liver Aorta Kidney Brain
CTL BetA PC
CREB SOD2
CREB
pCREB
SOD2
16.00 µm
16.00 µm
16.00 µm
AB
C
D
E
F
Fig. 7. BetA-mediated HBV inhibitory effect in mice through SOD2 suppression and ROS generation. HBV-transgenic mice were treated

tumor effect. Furthermore, this is the first time that
HBx has been found to be translocated into mitochon-
dria from the cytosol in BetA-treated HBV-infected
hepatocytes; given the fact that HBx could induce
apoptosis [33,34] and alter mitochondrial function by
inhibiting the mitochondrial electron transport chain
and oxidative phosphorylation (complexes I, III, IV,
and V) [31], we suppose that BetA-mediated HBx sup-
pression and translocation worsens the mitochondrial
dysfunction, which may further trigger apoptosis. As
SOD2 expression could totally abolish the BetA-
induced HBx suppression (Fig. 5A), we suppose that
HBx translocation to mitochondria could be associated
with BetA-induced SOD2 suppression. In order to
determine the possible role and effect of HBx in this
procedure, as shown in Fig. 5C–F, HBx protein was
overexpressed in WT hepatocytes overexpressing HBx,
and the related proapoptotic effect was measured. We
found that HBx alone (instead of full HBV infection)
showed a similar effect in WT cells after BetA treat-
ment, and caused small increases in caspase-3 activity
(Fig. 5C) and apoptosis rate (Fig. 5D), no increase in
SOD2 expression (Fig. 5E), and no HBx translocation
into mitochondria (data not shown). On the other
hand, in HBV-infected hepatocytes, SOD2 expression
was substantially increased, and became sensitive to
the BetA-induced proapoptotic effect, the HBx was
translocated into mitochondria, and the effect was nor-
malized by CREB overexpression (Fig. 5F), suggesting
that HBx alone does not directly contribute to BetA-

for subsequent deposition and detoxification as the
first target organ for xenobiotics, the high susceptibi-
lity of the liver could due to its higher SOD2 basal
expression and higher accessibility to BetA. On the
other hand, in brain tissue, the circulating BetA can
hardly gain access, owing to the protection of the
blood–brain barrier, so the brain is insensitive to BetA
treatment, even with higher SOD2 expression levels.
Taken together, these findings demonstrate that
BetA could achieve an impressive HBV-inhibitory
effect by specific suppression of SOD2 expression and
modulation of the mitochondrial redox balance. The
BetA used in this work was isolated and purified
from PC, a traditional Chinese herb that has been
succesfully used for the treatment of hepatitis B
patients, according to a secret recipe. According to the
Chinese traditional medicine theory: ‘Everything has
its own enemy from nature,’ which essentially means
D. Yao et al. Betulinic acid inhibits hepatitis B virus
FEBS Journal 276 (2009) 2599–2614 ª 2009 The Authors Journal compilation ª 2009 FEBS 2609
that everything within the world not only has its own
method of survival, but also has its own method of
destruction, thus preserving the balance of nature. In
fact, we can derive elements for good health from nat-
ure if we utilize it properly. Given the fact that PC is
well known and relatively nontoxic, with the promising
effects presented by its component BetA, PC scould be
a good candidate for anti-HBV drug development.
Experimental procedures
Materials and methods

SOD2, respectively. The CREB-1 cDNA was further sub-
cloned into the pGEX-4T vector (no. 27-4580-01; GE
Healthcare, Shanghai, China) for expression of CREB
protein in Escherichia coli BL21 cells, and this was fol-
lowed by glutathione S-transferase protein purification
with the MagneGST Protein Purification System
(no. V8600; Promega, Beijing, China) and thrombin prote-
ase digestion. The SOD2 promoter was amplified from
human genomic DNA and subcloned into the pGL3-basic
vector (no. E1751; Promega) to construct the SOD2
reporter plasmid. For mapping of the SOD2 promoter
response element, the related deletion or point mutation
constructs were generated by PCR methods or a site-
directed mutagenesis kit (no. Q9280; Promega). Detailed
information on those plasmids is available upon request.
Antibody against HBx (no. RD981038100) was obtained
from BioVendor Laboratories Ltd (Guangzhou, China),
and rabbit voltage-dependent anion selective channel
1 ⁄ porin antibody (V2139) was obtained from Sigma.
Antibodies against b-actin (sc-47778), CREB-1 (sc-58),
pCREB-1 (Ser133 phosphorylation, sc-7978) and SOD2
(sc-30080) were obtained from Santa Cruz (Beijing, China).
The mitochondrial and cytosolic fractions were pre-
pared and characterized using differential centrifugation
as previously described [39]. Protein concentrations were
measured with the BioRad protein assay kit (Bradford
method), according to the manufacturer’s instructions.
Small interfering RNAs for CREB-1 (no. s3491), SOD2
(no. 9052) and negative control (no. AM4636) were
obtained from Ambion (Shanghai, China). The SOD2

NaCl ⁄ P
i
, and 0.2 mL of 0.3 mgÆmL
)1
MTT solution was
then added at 25 °C for 3 h. Thereafter, the precipitated
blue formazan product was extracted by incubating sam-
ples with 0.1 mL of 10% SDS (dissolved in 0.01 m HCl)
overnight at 37 °C. The absorbances of formazan concen-
trations were determined at 570 nm, then normalized by
cell numbers and expressed as A ⁄ 10
6
cells.
Betulinic acid inhibits hepatitis B virus D. Yao et al.
2610 FEBS Journal 276 (2009) 2599–2614 ª 2009 The Authors Journal compilation ª 2009 FEBS
DNA synthesis evaluated by [
3
H]thymidine
incorporation
Cell proliferation was evaluated as the rate of DNA synthe-
sis by [
3
H]methylthymidine incorporation [41]. Cells were
pooled in 24-well plates up to 80% confluence, and differ-
ent concentrations of BetA were then added and incubated
for 48 h. At the end of the treatment, cells were incubated
with serum-free medium containing [
3
H]methylthymidine
(0.5 lCi per well) for 2 h, and then washed twice with

and quantitated by imagequant. The final results were
normalized to b-actin or porin (for mitochondrial protein).
Chromatin immunoprecipitation
Treated cells were crosslinked using 1% formaldehyde for
20 min and terminating with 0.1 m glycine. Cell lysates
were sonicated and centrifuged at 18 000 g for 12 min to
get supernatant. Five hundred micrograms of protein was
precleared by BSA ⁄ salmon sperm DNA plus preimmune
IgG and a slurry of protein A–agarose beads, as previously
described [42]. Immunoprecipitations were performed with
the indicated antibodies, BSA ⁄ salmon sperm DNA, and a
50% slurry of protein A–agarose beads. The immunopre-
cipitates were washed and eluted, and then incubated with
0.2 mgÆmL
)1
proteinase K for 2 h at 42 °C, and then for
6 h at 65 °C, to reverse the formaldehyde crosslinking.
DNA fragments were recovered by phenol ⁄ chloroform
extraction and ethanol precipitation. A 150 bp fragment
from the human SOD2 promoter was amplified by real-time
quantitative PCR.
In vitro phosphorylation of CREB at Ser133
Purified CREB was phosphorylated by the catalytic subunit
of PKA (no. P2645; Sigma) by incubating 2.0 lm CREB in
a reaction mixture containing 4.0 lm ATP (or UTP for
mock reactions), 8 mm MgCl
2
and 100 U of PKA in
25 mm NaCl ⁄ P
i

Evaluation of apoptosis
Apoptosis was evaluated by TUNEL assay using an In Situ
Cell Death Detection Kit (Roche, Shanghai, China). Cells
were fixed in 4% paraformaldehyde, and labeled with
TUNEL reagents. Stained cells were photographed with a
fluorescence microscope, and further quantified by fluores-
cence activated cell sorting analysis. Caspase-3 activity was
determined with an ApoAlert caspase assay kit (Clontech,
Beijing, China). Treated cells were harvested, and 50 lgof
protein was incubated with the fluorogenic peptide substrate
Ac-DEVD-7-amino-4-trifluoromethyl coumarin. The initial
rate of free Ac-DEVD-7-amino-4-trifluoromethyl coumarin
release was measured using an FLx800 microplate reader
(Bio-Tek, Guangzhou, China) at excitation ⁄ emission wave-
lengths of 380 ⁄ 505 nm, and enzyme activity was calculated
as pmol ⁄ min ⁄ mg or as arbitrary units [39].
Measurement of oxidative stress
Intracellular ROS generation was determined by using oxi-
dation of 2¢,7¢-dihydrochlorofluorescein-diacetate. Treated
D. Yao et al. Betulinic acid inhibits hepatitis B virus
FEBS Journal 276 (2009) 2599–2614 ª 2009 The Authors Journal compilation ª 2009 FEBS 2611
cells were washed and incubated with 0.1 mL of 10 lm
2¢,7¢-dihydrochlorofluorescein-diacetate, and the fluorescence
was measured at excitation ⁄ emission wavelengths of 485 ⁄
530 nm, using an FLx800 microplate fluorescence reader
(Bio-Tek). The data were normalized to arbitrary units [39].
Measurement of HBV replication
HBV-infected hepatocytes were seeded in 24-well plates,
and then subjected to BetA treatment for 48 h. Cell num-
bers were determined by Trypan blue exclusion. Secretion

[39]. The in vitro staining of superoxide anions (O
2
·
)
) was
performed with the oxidative fluorescent dye dihydroethi-
dium [46,47]. Briefly, fresh and unfixed liver tissues were
frozen and cut in a cryostat into 30 lm sections and
placed on glass slides. Samples were then incubated at
room temperature for 30 min with dihydroethidium
(0.002 mmolÆL
)1
) and protected from light. Images were
obtained using a laser (krypton ⁄ argon) scanning confocal
microscope with fluorescence excitation ⁄ emission at
488 ⁄ 610 nm.
Statistical analysis
Data are given as mean ± standard deviation. All experi-
ments were performed at least three times. All analyses
were performed using spss 15.0 statistical software. Stu-
dent’s unpaired t-test or ANOVA were used to determine
the statistical significance of different groups. A P-value
< 0.05 was considered to be significant.
Acknowledgements
This work was supported by the National Natural Sci-
ence Foundation of China (Project No. 30270563) and
the Science Research Foundation of Health Depart-
ment of Hubei Province (Project No. JX2A04).
References
1 Lau JY & Wright TL (1993) Molecular virology and

human immunodeficiency virus type 1 specific inhibitors
with a new mode of action. J Med Chem 39, 1056–1068.
9 Yogeeswari P & Sriram D (2005) Betulinic acid and its
derivatives: a review on their biological properties. Curr
Med Chem 12, 657–666.
10 Fulda S, Scaffidi C, Susin SA, Krammer PH, Kroemer
G, Peter ME & Debatin KM (1998) Activation of mito-
chondria and release of mitochondrial apoptogenic fac-
tors by betulinic acid. J Biol Chem 273, 33942–33948.
11 Yen HC, Oberley TD, Vichitbandha S, Ho YS & St
Clair DK (1996) The protective role of manganese
Betulinic acid inhibits hepatitis B virus D. Yao et al.
2612 FEBS Journal 276 (2009) 2599–2614 ª 2009 The Authors Journal compilation ª 2009 FEBS
superoxide dismutase against adriamycin-induced acute
cardiac toxicity in transgenic mice. J Clin Invest 98,
1253–1260.
12 Lam EW, Zwacka R, Seftor EA, Nieva DR, Davidson
BL, Engelhardt JF, Hendrix MJ & Oberley LW (1999)
Effects of antioxidant enzyme overexpression on the
invasive phenotype of hamster cheek pouch carcinoma
cells. Free Radic Biol Med 27, 572–579.
13 Xu Y, Fang F, Dhar SK, St Clair WH, Kasarskis EJ &
St Clair DK (2007) The role of a single-stranded nucle-
otide loop in transcriptional regulation of the human
sod2 gene. J Biol Chem 282, 15981–15994.
14 Xu Y, Krishnan A, Wan XS, Majima H, Yeh CC,
Ludewig G, Kasarskis EJ & St Clair DK (1999)
Mutations in the promoter reveal a cause for the
reduced expression of the human manganese super-
oxide dismutase gene in cancer cells. Oncogene 18,

tissues. Proc Natl Acad Sci USA 102, 4459–4464.
22 Vallejo M, Gosse ME, Beckman W & Habener JF
(1995) Impaired cyclic AMP-dependent phosphorylation
renders CREB a repressor of C ⁄ EBP-induced transcrip-
tion of the somatostatin gene in an insulinoma cell line.
Mol Cell Biol 15, 415–424.
23 Bito H, Deisseroth K & Tsien RW (1996) CREB phos-
phorylation and dephosphorylation: a Ca(2+)- and
stimulus duration-dependent switch for hippocampal
gene expression. Cell 87, 1203–1214.
24 Costantini P, Jacotot E, Decaudin D & Kroemer G
(2000) Mitochondrion as a novel target of
anticancer chemotherapy. J Natl Cancer Inst 92,
1042–1053.
25 Lamph WW, Dwarki VJ, Ofir R, Montminy M &
Verma IM (1990) Negative and positive regulation by
transcription factor cAMP response element-binding
protein is modulated by phosphorylation. Proc Natl
Acad Sci USA 87, 4320–4324.
26 Sharma N, Lopez DI & Nyborg JK (2007) DNA bind-
ing and phosphorylation induce conformational altera-
tions in the kinase-inducible domain of CREB.
Implications for the mechanism of transcription func-
tion. J Biol Chem 282, 19872–19883.
27 Johannessen M, Delghandi MP, Rykx A, Dragset M,
Vandenheede JR, Van Lint J & Moens U (2007) Protein
kinase D induces transcription through direct phosphor-
ylation of the cAMP-response element-binding protein.
J Biol Chem 282, 14777–14787.
28 Chrivia JC, Kwok RP, Lamb N, Hagiwara M, Montmi-

36 Guidotti LG, Matzke B, Schaller H & Chisari FV
(1995) High-level hepatitis B virus replication in trans-
genic mice. J Virol 69 , 6158–6169.
D. Yao et al. Betulinic acid inhibits hepatitis B virus
FEBS Journal 276 (2009) 2599–2614 ª 2009 The Authors Journal compilation ª 2009 FEBS 2613
37 Chisari FV (1995) Hepatitis B virus transgenic mice:
insights into the virus and the disease. Hepatology 22,
1316–1325.
38 Patel RD, Hollingshead BD, Omiecinski CJ & Perdew
GH (2007) Aryl-hydrocarbon receptor activation regu-
lates constitutive androstane receptor levels in murine
and human liver. Hepatology 46, 209–218.
39 Yao D, Shi W, Gou Y, Zhou X, Yee Aw T, Zhou Y &
Liu Z (2005) Fatty acid-mediated intracellular iron
translocation: a synergistic mechanism of oxidative
injury. Free Radic Biol Med 39, 1385–1398.
40 Cheng X, Shin YG, Levine BS, Smith AC, Tomaszew-
ski JE & van Breemen RB (2003) Quantitative analysis
of betulinic acid in mouse, rat and dog plasma using
electrospray liquid chromatography ⁄ mass spectrometry.
Rapid Commun Mass Spectrom 17, 2089–2092.
41 Somasundaram K & El-Deiry WS (1997) Inhibition of
p53-mediated transactivation and cell cycle arrest by
E1A through its p300 ⁄ CBP-interacting region. Oncogene
14, 1047–1057.
42 Metivier R, Penot G, Hubner MR, Reid G, Brand H,
Kos M & Gannon F (2003) Estrogen receptor-alpha
directs ordered, cyclical, and combinatorial recruitment
of cofactors on a natural target promoter. Cell 115,
751–763.