Hepatitis B virus X (HBx) play an anti-apoptosis role in hepaticprogenitor cells by activating Wnt/b-catenin pathway

Lihong Shen • Xifeng Zhang • Daixi Hu •

Tao Feng • Hongli Li • Yongliang Lu •

Jiayi Huang

Received: 10 April 2013 / Accepted: 2 August 2013 / Published online: 10 August 2013

� Springer Science+Business Media New York 2013

Abstract Increasing evidence has shown that normal

stem cells may act as cancer-initiating cells and contribute

to the development and progression of cancer. HBx has a

close relationship with hepatocellular carcinoma, however,

the role of HBx in hepatic progenitor cells (HPCs) is poorly

understood. In this study, we sought to determine the role

of HBx in regulating HPCs apoptosis and the underlying

molecular mechanism(s) using HPCs derived from mouse

fetal liver. The apoptotic ratio of HPCs infected with

adenovirus-expressing HBx (Ad-HBx) was examined using

flow cytometry. Results showed that the Ad-HBx treatment

led to substantially decreased apoptotic ratio of HPCs, as

confirmed by the Hoechst 33342 staining and terminal

deoxynucleotidyl transferase-mediated dUTP nick end-

labeling (TUNEL). Possible alterations of relative proteins

were examined using Western blot and real-time PCR

assays. The HBx expression in HPCs increased the

expression levels of Bcl2 and Mcl1 while decreasing the

expression levels of Bax and cleaved caspase-9 and -3. In

addition, the mRNA and protein expression levels of

b-catenin were both increased. The b-catenin protein were

mainly accumulated in cytoplasm and tended to transfer

into cell nucleus after Ad-HBx treatment. The over-

expression of b-catenin decreased the apoptotic ratio of

HPCs and inhibited the expression of cleaved caspase-9

and -3 while blocking b-catenin expression resulted in the

opposite results. Taken together, our results strongly sug-

gested that the HBx protein may inhibits apoptosis of

hepatic progenitor cells, at least in part by activating the

WNT/b-catenin pathway. This provided a new insight into

the molecular mechanism of HBx-mediated live

carcinogenesis.

Keywords Hepatitis B virus X � Hepatic progenitor

cells � Apoptosis � WNT/b-catenin pathway � Cancer

stem cells

Introduction

Cancer stem cells are defined as ‘‘a small subset of cancer

cells within a cancer that constitute a reservoir of self-

sustaining cells with the exclusive ability to self-renew and

to cause the heterogeneous lineages of cancer cells that

comprise the tumor’’ [1]. The first convincing evidence for

cancer stem cells was demonstrated in acute myelogenous

leukemia (AML) in 1994 by Dick and co-workers [2, 3].

And the first solid tumor stem cells, breast cancer stem

cells, were reported by Clarke et al. [4]. Since then, cancer

stem cells draw much attention and have been widely

studied and characterized in many different types of human

tumors, including brain tumors [5], multiple myeloma [6],

colon cancer [7], prostate cancer [8], head and neck cancer

[9], melanoma [10], hepatocellular carcinoma (HCC) [11],

pancreatic cancer [12], and lung cancer [13]. Cancer and

Lihong Shen and Xifeng Zhang contributed equally to the work.

L. Shen � X. Zhang � D. Hu � T. Feng � H. Li � Y. Lu � J. Huang

Molecular Medicine and Cancer Research Center,

Chongqing Medical University, Chongqing 400016,

People’s Republic of China

L. Shen � X. Zhang � D. Hu � T. Feng � H. Li � Y. Lu

Departments of Biochemistry and Molecular Biology,

Chongqing Medical University, Chongqing 400016,

People’s Republic of China

J. Huang (&)

Departments of Pathophysiology, Chongqing Medical

University, Chongqing 400016, People’s Republic of China

e-mail: e738741@126.com

123

Mol Cell Biochem (2013) 383:213–222

DOI 10.1007/s11010-013-1769-5

normal stem cells have same properties, such as differen-

tiation and self-renewal and some types of cancer stem

cells and stem cells share tightly regulated self-renewal

pathways, including Notch pathway [14], Wnt pathway

[15], and sonic hedgehog (Shh) pathway [16]. So, cancer

stem cells could originate from normal stem cells or pro-

genitor cells after acquisition of multiple mutations [17].

On the other hand, cancer stem cells could also be derived

from mature cells that have undergone a de-differentiation

or a transdifferentiation process (by cell fusion and hori-

zontal gene-transfer) [18], both genetic and epigenetic

factors could account for this transformation [18].

The hepatitis B virus (HBV) is the smallest DNA virus-

infecting humans and the prototype member of the hepa-

dnaviridae family [19]. The relationship of HBV infection

to primary liver cancer is based on robust epidemiologic

evidence: liver cancer frequently occurs in HBV-endemic

areas; the rate of chronic HBV carriers is higher among

liver cancer patients and the risk of developing hepatic

tumors is substantially increased in HBV carriers than in

the general population. In the occurrence and progression

of HBV-related HCC, the HBV-encoded X protein (HBx)

is considered to be a key component [20]. HBx is a protein

of Mr 17,000, whose structural and biochemical properties

are largely unknown. The most extensively studied prop-

erty of HBx is its capacity to trans-activate. This activity is

believed to be crucial for the development of liver cancer,

because it is involved in HBV transcription and replication

[21], up-regulating many genes that mediate oncogenesis,

proliferation, and immune responses [22]. Although the

role of HBx in the occurrence of HBV-related HCC has

been intensively studied, the underlying mechanism is still

not clear.

Cancer stem cell theory give a new sight for the study of

HBx. Cancer stem cells also be found in HCC [11], they

arise from dedifferentiation of mature hepatocytes or arrest

maturation of determined stem cells [23]. It is reported that

HepG2 cells stably transduced with HBx highly express

Oct-4, Nanog, Klf-4, b-catenin, and epithelial cell adhesion

molecule (EpCAM) in vitro and in vivo. Moreover, HBx-

stimulated cell migration, growth in soft agar, and spheroid

formation. It means that HBx could promote the dediffer-

entiation of mature hepatocytes and the appearance of liver

cancer stem cells, contributing to hepatocarcinogenesis

[24]. In L02 cells, stable HBx transfection also resulted in a

malignant phenotype [25]. However, whether HBx could

induce hepatic stem cell maturation arrest is known little.

In 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-trea-

ted HBx transgenic mice, elevated number of EpCAM(?)

cells with characteristics of HPCs were observed, and all

HBx transgenic mice developed liver tumors characterized

with histological features of both HCC and cholangiocar-

cinoma after 7 months of DDC feeding. This indicated that

HBx may induce malignant transformation of HPCs that

contributes to tumorigenesis [26]. In our previous study,

using HPCs derived from the E14.5 mouse fetal liver, we

directly observed that HBx could inhibited the terminal

hepatic differentiation, leading to an increase in the

S-phase cell cycle fraction and a decrease in the apoptotic

ratio [27]. In this study, using flow cytometry, Hoechst

33342 staining and TUNEL, the anti-apoptosis effection of

HBx on HPCs was confirmed. At the same time, Wnt/b-

catenin pathway was activated. After infected with Ad-b-

catenin, the ratio of apoptosis in HPCs decreased and the

activities of caspase-9 and -3 were inhibited. The results

demonstrated that the HBx inhibited the apoptosis of HPCs

by activating the Wnt/b-catenin pathway. It gives a new

insight in understanding the role of HBx in HBV-related

liver oncogenesis and development.

Materials and methods

Antibodies and reagents

Monoclonal anti-HBx antibody was purchased from

Chemicon (Temecula, CA, USA). Rabbit polyclonal anti-

Bcl2, anti-Bax, anti-caspase 3 and 9 antibodies were pur-

chased from bioword (Louis Park, MN, USA). Rabbit

polyclonal anti-Mcl1 antibody was purchased from Bioleg-

end (San Diego, CA, USA). Rabbit polyclonal anti-cleaved

caspase-3 and -9 antibodies were purchased from Cell Sig-

naling Inc. (Danvers, MA, USA). Monoclonal anti-actin

antibody, goat anti-mouse IgG-AP, goat anti-rabbit IgG-AP,

BCA protein assay kit, BCIP/NBT alkaline phosphatase

color development Kit, the BeyoECL Plus Western blotting

detection system, Hoechst33342 staining and terminal

deoxynucleotidyl transferase-mediated dUTP nick end-

labeling (TUNEL) were purchased from Beyotime Institute

of Biotechnology (Jiangsu, China). Primers were synthe-

sized by Shanghai Sangon Biotech (Shanghai, China). All

other chemicals and reagents were of analytical grade.

Cell culture and viral infection

Recombinant adenoviruses-expressing green fluorescent pro-

tein (Ad-GFP), HBx (Ad-HBx), and b-catenin (Ad-b-catenin)

were provided by Dr. T. -C (University of Chicago Medical

Center). HEK-293 cell line was obtained from the American

Type Culture Collection (Manassas, VA, USA). The HPCs

(HP14.5) were isolated from the E14.5 mouse fetal liver and

infected with a retrovirus packaged SSR No. 69, which

employed the overexpression of SV40 large T antigen flanked

with loxP sites, to establish reversible stable progenitor lines.

We selected the single clone cell expressing early marker and

late markers and induced the cells to differentiate into hepatic

214 Mol Cell Biochem (2013) 383:213–222

123

and bile duct cells designated as HP14.5 [28, 29]. All cell lines

were cultured in the Dulbecco’s Modified Eagle’s Medium

(DMEM; Hyclone) supplemented with 10 % fetal bovine

serum (FBS; Hyclone), 100 IU/ml penicillin, and 100 mg/ml

streptomycin. Cells were maintained at 37 �C in a humidified

atmosphere of 5 % CO2. Cells at 60–70 % confluency were

infected with recombinant Ad vectors (30 PFU/cell) in phos-

phate-buffered saline (PBS) at 37 �C for 1 h with occasional

rotation. The cells were incubated in fresh DMEM—10 %

FBS before analysis.

Reverse-transcription PCR (RT-PCR) and quantitative

real-time PCR (qPCR)

Total RNA was extracted from cells using TRIzol reagent

(Invitrogen, CA, USA) following the manufacturer’s

instructions. RNA yield and purity were tested by UV

absorbance spectroscopy. Total RNA (1 lg) was reverse-

transcribed in 20 ll reactions using the cDNA Synthesis Kit

(TaKaRa). The cDNA products were used as template for

semi-quantitative RT-PCR and qPCR. Semi-quantitative RT-

PCR reactions (15 ll) were prepared using the PCR reagent

kit (TakaRa), and the PCR amplification conditions were as

follows: initial 94 �C for 3 min; 30 cycles of 94 �C for 30 s,

55 �C for 30 s, and 72 �C for 30 s; followed by 72 �C for

5 min. The GAPDH mRNA expression was examined as an

internal control under the same RT-PCR conditions. The PCR

products were separated by 2.0 % agarose gel electrophoresis

and the gel images were photographed with a digital camera

system. qPCR reactions (20 ll) were prepared using the

SYBRGreen PCR Master Mix reagent kit (TaKaRa), and the

PCR amplification was carried out on an ABI 7500 real-time

PCR system (USA) under the following thermal cycling

conditions: 50 �C for 2 min, 95 �C for 10 min, and 40 cycles

of 95 �C for 5 s, 60 �C for 15 s, and 72 �C for 15 s. Relative

expression of HBx was calculated using the 2-44ct method.

The sequences of PCR primers used for both RT-PCR and

real-time PCR were as follows: HBx forward, 50-cggaattcc-

gatggctgctaggctgtg-30; HBx reverse, 50-ccctcgaggggttgcat

ggtgctggt-30; Bcl2 forward, 50-atctccctgttgacgctct-30; Bcl2

reverse, 50-catcttctccttccagcct-30; Mcl1 forward, 50-gtcccgtt

tcgtccttacaa-30; Mcl1 reverse, 50-gctccggaaactggacatta-30;Bax forward, 50-tgcagaggatgattgctgac-30; Bax reverse, 50-gatc

agctcgggcactttag-30; b-catenin forward, 50-caatcaagagagcaag

ctcatc-30; b-catenin reverse, 50-agtcgctgacttgggtctgt-30;GAPDH forward, 50-accacagtccatgccatcac-30; GAPDH

reverse, 50-tccaccaccctgttgctgat-30.

Flow cytometry (FCM) assay

The HPCs were cultured on 6-well plates for 72 h post-

infection with Ad-GFP, Ad-HBX, or Ad-b-catenin. Cells

were synchronized using a serum-free medium for 24 h and

then stimulated by supplemented with a complete medium.

The number of apoptotic cells were measured at an early

growth phase using Annexin V-PE and 7-amino-actino-

mycin D (7-AAD).

Morphological studies of apoptotic cell

The HPCs were cultured on 24-well plates for 72 h post-

infection with Ad-GFP, Ad-HBX, or Ad-b-catenin, and then

fixed with 4 % paraformaldehyde for 30 min at 25 �C. The

preparations were washed with cold PBS three times and cells

were stained with the Hoechst 33342 dye (Beyotime Inst.

Biotech) for 15 min at room temperature. Then, cells were

examined under a fluorescent microscope with an excitation

wavelength of 365 nm. The cells with condensed chromatin

and shrunken nuclei were counted as apoptotic cells.

TUNEL staining

The TUNEL method was used to label the 30-end of DNA

fragments in apoptotic HPCs. The infected HPCs were

plated on glass coverslips, rinsed with PBS, and fixed with

4 % paraform in PBS. Then, the HPCs were rinsed with

PBS and permeabilized with 0.1 % Triton X-100 for FITC

end-labeling of DNA fragments in apoptotic HPCs using

the TUNEL cell apoptosis detection kit. The FITC-labeled

TUNEL-positive cells were imaged under a fluorescent

microscope using 488-nm excitation and 530-nm emission.

Immunocytochemistry

Seventy-two hours post-infection, the cells were fixed with

4 % paraformaldehyde, permeabilized in 0.5 % Triton

X-100 in PBS and then incubated in 0.3 % H2O2 for 5 min.

Thereafter, the sections were washed twice with PBS,

blocked with normal goat serum, and incubated with b-

catenin antibody (1:50, Bioword Biotechnology, CA, USA)

overnight at 4 �C. The cells were stained using diam-

inobenzidine (DAB, Dako, Carpinteria, CA, USA).

Preparation of cell extracts and Western blot analysis

The whole cellular lysates were prepared with the radio-

immunoprecipitation assay (RIPA) lysis buffer, and the

cytosolic and nuclear fractions were separated according to

the manufacturer’s instruction (Pierce, Rockford, IL,

USA). Cellular extracts (40 lg) were separated by 6–12 %

SDS-PAGE and transferred to polyvinylidene fluoride

(PVDF) membranes. The blots were probed with various

antibodies: Bcl2 (1:500), Mcl1 (1:500), Bax (1:500), b-

actin (1:500), caspase3 (1:250), caspase9 (1:250), cleaved

caspase-3 (1:250), cleaved caspase-9 (1:250), and b-cate-

nin antibodies (1:500). To visualize the antibody-bound

Mol Cell Biochem (2013) 383:213–222 215

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protein, appropriate secondary antibodies (1:1,000) and

ECL detection solutions (Pierce) were applied. The scan-

ned images were quantified using Quantity-One software

(BioRad, Hercules, CA, USA).

Statistical analysis

Standard deviation (SD) of replicate data was calculated

using the Microsoft Excel program. Statistically significant

differences between samples were evaluated using the two-

tailed Student’s t test.

Results

Expression of hepatitis B virus X protein in hepatic

progenitor cells

To investigate the potential apoptotic ability of HBx in

hepatic progenitor cells, we first prepared adenovirus

expression HBx. We infected hepatic progenitor cells

HP14.5 with Ad-HBx and confirmed the expression of HBx

by RT-PCR and Western-blot. The results showed that

HBx was positively expressed in the HPCs infected with

Ad-HBx (Fig. 1a, b).

Inhibitory effects of hepatitis B virus X protein

on apoptosis in HPCs

The relationship between HBx and apoptosis is a topic of

HBV biology that illustrates its complex and paradoxical

effects. Several studies have demonstrated that HBx could

inhibit apoptosis by sequestering p53 in the cytoplasm,

resulting in the activation of the PI3K pathway and up-

regulation of the SAPK/JNK pathway [30–32]. It has also

been found pro-apoptotic by prolonging the stimulation of

the N-myc and MEKK1 pathways via interactions with

c-FLIP and hyper-activation of caspase-8 and -3, respec-

tively [33, 34]. In our study, HPCs were divided into mock

group and HBx group, which infected with Ad-GFP and

Ad-HBx, respectively, and their ratio of apoptosis was

monitored by TUNEL staining, Hoechst 33342 staining

and flow cytometry.

In situ TUNEL staining, the TUNEL-negative cells had

well-distributed green staining and TUNEL-positive cells

had highly condensed, brightly green staining. From the

results we can see that in HBx group, the TUNEL-positive

cells clearly decreased (Fig. 2a). Hoechst 33342 staining

showed that the number of apoptotic cells with reduced cell

sizes and increased nuclear chromatin condensation were

also decreased after infected with Ad-HBx (Fig. 2b). Cells

treated with HBx for 48 h were harvested and stained with

Annexin V-PE and 7-AAD. Flow cytometry was used to

evaluate the apoptotic cells at the early phase. After treated

with HBx, the apoptotic cells clearly decreased in HP14.5

cells compared with cells infected with Ad-GFP (Fig. 2c).

These results indicated that the high expression of HBx

inhibited the apoptosis of HP14.5 cells.

To reveal the mechanism for the decreased apoptotic

ratio after the HBx treatment, we searched for possible

alterations of apoptotic regulators such as Bcl-2, Bax and

Mcl1. RT-PCR and Western blot assays showed that 72-h

post-infection with HBx, the mRNA and protein expression

levels of Bax were decreased, while those of Bcl-2 and

Mcl1 were increased in HP14.5 cells (Fig. 3a, b). In

addition, Western blot assay showed that the activities of

caspase-9 and -3 were decreased in HBx-infected HP14.5

cells (Fig. 3c). It was likely that the HBx-inhibited cell

apoptosis by disturbing the balance of Bcl2 family protein

and inhibiting the activities of caspase-9 and -3.

Hepatitis B virus X protein activated Wnt/b-catenin

signaling pathways in HPCs

Wnt/b-catenin signaling pathway is known to be respon-

sible for activation and transformation of stem/progenitor

cells [35–37]. To identify whether this pathway was

involved in inhibited apoptosis of HPCs treated with HBx,

we detected activity of Wnt/b-catenin signaling pathway in

HPC cells expressing HBx. As shown in Fig. 2d, b-catenin

mRNA and protein expression levels were both increased

in HPC cells by HBx. Using immunohistochemical label-

ing, we observed that the b-catenin proteins were mainly

accumulated in cytoplasm and tended to transfer into cell

nucleus (Fig. 2e). These results indicated that the expres-

sion of HBx protein activated the Wnt/b-catenin pathway

in HPC cells, and the activation of Wnt/b-catenin pathway

may be necessary for the inhibited apoptosis of HPCs.

b-Catenin over-expression reduced the apoptosis

of HPCs

To study the effection of activated Wnt/b-catenin pathway

on apoptosis of HPC cells, we prepared adenovirus

Fig. 1 RT-PCR and West blot assays of hepatitis B virus X gene

expression in HP14.5 cells 72 h post-infection. HP14.5 cells were

infected with Ad-HBx or Ad-GFP. All samples were normalized to

GAPDH in RT-PCR assay and b-actin in Western blot assay. The

expression of HBx mRNA and protein in HP14.5 cells were

determined by RT-PCR (a) and Western blotting (b) 72 h post-

infection

216 Mol Cell Biochem (2013) 383:213–222

123

expression b-catenin. We infected hepatic progenitor cells

HP14.5 with Ad-b-catenin and monitor the change of

apoptosis ratio by using TUNEL staining, Hoechst 33342

staining and flow cytometry.

From the results we could see that after infected with

Ad-b-catenin, the TUNEL-positive cells clearly decreased

(Fig. 4a), and Hoechst 33342 staining had the similar

results (Fig. 4b). Cells treated with b-catenin for 48 h were

harvested and stained with Annexin V-PE and 7-AAD.

FCM showed that the radio of apoptotic cells dropped from

11.55 % in mock group to 4.42 % in b-catenin group

(Fig. 4c). Altogether, these results indicated that the over

expression of b-catenin inhibited the apoptosis of HP14.5

cells.

We also detected the possible alterations of apoptotic

regulators. Western blot assays showed that activated

Fig. 2 Anti-apoptosis of HP

14.5 cells induced by hepatitis B

virus X protein and activating

Wnt/b-catenin pathway. HP14.5

cells were infected with Ad-

HBx or Ad-GFP (negative

control). Cells were examined

by TUNEL staining (a) (9100),

Hoechst 33342 staining

(b) (9200) and FCM (c). Yellow

arrows indicate apoptotic

nuclei. Apoptosis ratio

(%) = (apoptotic cells/total

cells) 9 100 %. The mRNA

and protein expression levels of

b-catenin were determined by

RT-PCR and Western blot

assays (d).

Immunocytochemistry was used

to identify the effect of HBX on

the distribution of b-catenin (e).

(Color figure online)

Mol Cell Biochem (2013) 383:213–222 217

123

Wnt/b-catenin pathway inhibited the activities of caspase-

9 and -3, but had no effect on the protein expression of

Bcl-2 and Bax (Fig. 5). These indicated that the HBx-

inhibited cell apoptosis by activating the Wnt/b-catenin

signaling pathway, which suppressed the activities of

caspase-9 and -3.

Fig. 3 The mRNA and protein expression of Bax, Bcl-2 and Mcl1,

and the detection of activated caspases in HP14.5 cells induced by

hepatitis B virus X protein. HP14.5 cells were infected with Ad-HBx

or Ad-GFP (negative control). The mRNA (a) and protein (b) expres-

sion levels of Bax, Bcl-2, Mcl1 were determined by RT-PCR and

Western blot assays 72 h post-infection. The enzyme activities of

caspase-3 and -9 (c) were analyzed by Western blot assay. Data are

expressed as mean ± SD (n = 3), *P \ 0.05 versus Ad-GFP

treatment

Fig. 4 b-Catenin over-

expression induced anti-

apoptosis in HPCs. HP14.5 cells

were infected with Ad-b-catenin

or Ad-GFP (negative control).

Apoptotic cells were examined

by TUNEL staining (9100) a,

Hoechst 33342 staining (9200)

b and FCM c, Yellow arrows

indicate apoptotic nuclei.

Apoptosis ratio

(%) = (apoptotic cells/total

cells) 9 100 %. (Color figure

online)

218 Mol Cell Biochem (2013) 383:213–222

123

b-Catenin blocking neutralized the anti-apoptosis

effection of HBx in HPCs

To conform the effection of Wnt/b-catenin pathway acti-

vated by HBx on apoptosis of HPC cells, we also con-

structed b-catenin knockdown vectors and the efficiency of

Ad-sib-catenin was confirmed by RT-PCR and Western-

blot (Fig. 6a). We co-infected hepatic progenitor cells

HP14.5 with Ad-HBx and Ad-si-b-catenin, and monitored

whether b-catenin blocking could neutralize the anti-

apoptosis effection of HBx in HPCs.

From the results we can see that, comparing with HBx

group, co-infected HPCs with Ad-HBx and Ad-si-b-catenin

could result in Hoechst-positive cells clearly increased

(Fig. 6b). RT-PCR and Western blot assays showed that

the mRNA and protein expression levels of Bax were

increased, while those of Bcl-2 and Mcl1 were decreased in

HP14.5 cells co-infected with Ad-HBx and Ad-si-b-catenin

(Fig. 6c, d). At the same time, the activities of caspase-3

and -9 were also increased, confirmed by RT-PCR and

Western blot assays (Fig. 6c, d). Altogether, these results

indicated that the blocking of b-catenin expression could

induce the apoptosis of HP14.5 cells.

Discussion

The relationship between HBx and apoptosis has been

intensively studied. Various studies have proposed that HBx

prevents apoptosis by interfering with pathways that acti-

vated by Fas and TGF-b [32, 38] or by interacting directly

with p53 [39]. In serum-deprived or in pro-apoptotic drug-

treated Chang cells transiently transfected with HBx could

result in apoptosis inhibited [40, 41]. Similarly, in stably

transfected Hep3B cells, HBx inhibits TGF-b-induced

apoptosis [38] or caspase-3 [41]. In another anti-apoptotic

mechanism, HBx decreases caspase activity through its

association with survivin, an anti-apoptotic protein that is

overexpressed in most human cancers [41]. In contrast to the

proposed anti-apoptotic functions of HBx, several reports

have observed increased sensitivity to pro-apoptotic stimuli

following mitochondrial damage by HBx [42, 43]. Cyto-

chrome C release and mitochondrial aggregation have been

proposed as mechanisms of these effects [43–45]. These

conflict results illustrates its complex and paradoxical

effects and indicate that under different conditions, the HBx

may play various roles in mediating cell apoptosis during

the occurrence and progression of HBV-related HCC.

The effect of HBx on apoptosis of HPCs, liver cancer

trigger cells, are worth to study. This study investigated

the regulatory mechanism of anti-apoptotic function of

HBx in HPCs. We established reversible stable HPCs

derived from the E14.5 mouse fetal liver through the

retroviral integration of SV40 large T cells (designated as

HP14.5 cells). The HP14.5 cells were shown to express

high levels of early liver stem cell markers (e.g., Oct-3/4,

DLK, and c-kit) with low levels of late liver markers (e.g.,

ALB and UGT1A). Compared with negative control, the

HBx-infected HPCs exhibited less apoptotic nuclear con-

densation and a lower rate of cell death. This could be

related to several studies which demonstrated that the

HBx prevented apoptosis by interfering with cellular

proteins involved in the CD95- and transforming growth

factor b (TGF-b)-mediated apoptosis pathways [32, 46,

47], direct interaction with p53 [39] and caspase-3 [48], or

enhancement of MAT2A [49] and caspase-independent

pathway [50]. In addition, the expression levels of anti-

apoptosis-related proteins were found remarkably up-reg-

ulated while the pro-apoptosis was significantly down-

regulated in Ad-HBx-infected cells. Taken together, these

results indicate that in HPC cells, HBx could inhibited its

apoptosis by disturbing the balanced expression of Bcl2

family-related proteins and the caspase protein, which

maybe an underlying mechanism in HBx inducing

malignant transformation of HPCs.

The Wnt/b-catenin pathway has long been considered

involved in embryonic liver development and hepatocar-

cinogenesis [51, 52]. The b-catenin is a key component of

Wnt signaling pathway and its translocation to the nucleus

initiates transcription of downstream target genes [53]. b-

catenin can bind to T-cell factor/lymphoid-enhancing fac-

tor (Tcf/Lef) in the nucleus and acts as a co-activator to

stimulate the transcription of target genes such as c-myc

and cyclinD1 [54]. The activation of b-catenin/T cell factor

(Tcf)1-mediated transcription by Wnt signal transduction is

of great importance to its biological function [55].

Abnormal activation of b-catenin is considered to be a

strong driving force in hepatocellular carcinogenesis. In

this study, we proposed that the HBx protein acted as the

Fig. 5 Effect of activating Wnt/b-catenin signaling on Bcl2, Bax,

Mcl1, and activity of caspase in Ad-b-catenin-infected HPCs. HP14.5

cells were infected with Ad-b-catenin or Ad-GFP (negative control).

The Bcl2, Bax, Mcl1 and enzyme activities of caspase-3 and -9 were

analyzed by Western blot assay. Data are expressed as mean ± SD

(n = 3), *P \ 0.05 versus Ad-GFP treatment

Mol Cell Biochem (2013) 383:213–222 219

123

stressor to activate the Wnt/b-catenin signaling pathway

and suppressed the apoptosis in HPCs by regulating the

caspase expression.

After HBx infection, the expression levels of b-catenin in

HPCs were obviously increased and the b-catenin proteins

were mainly accumulated in cytoplasm, which tended to

transfer into cell nucleus. FCM and Western blot assays

showed that the HPCs infected with Ad-b-catenina had a

decreased apoptotic rate and remarkably downregulated

expressions levels of cleaved caspase-3 and cleaved caspase-

9 proteins. This indicated that the HBx-inhibited apoptosis by

activating the Wnt/b-catenin pathway, which suppressed the

caspase-dependent apoptosis, and that the anti-apoptosis

induced by HBx referred to the mitochondrial pathway.

Nevertheless, further study is needed to investigate the effects

of activating Wnt/b-catenin pathway on apoptosis.

Fig. 6 b-Catenin blocking

neutralized the anti-apoptosis

effection of HBx in HPCs.

HP14.5 cells were treated with

HBx alone or HBx plus si-b-

catenin. a The change of b-

catenin expression was

confirmed by RT-PCR and

Western blot. Data are

expressed as mean ± SD

(n = 3), *P \ 0.05 versus Ad-

HBx treatment. b Apoptotic

cells were examined by Hoechst

33342 staining (9200). Yellow

arrows indicate apoptotic

nuclei. c Apoptotic-relation

genes were analyzed by Q-PCR.

Data are expressed as

mean ± SD (n = 3), *P \ 0.05

versus Ad-HBx treatment.

d Apoptotic-relation genes were

analyzed by Western blot assay.

The results were confirmed in at

least three batches of

independent experiments.

(Color figure online)

220 Mol Cell Biochem (2013) 383:213–222

123

Conclusions

In summary, we have demonstrated that HBx treatment

could reduce the apoptotic ratio of HP14.5 cells, which is

thought as the cancer trigger cells. It might be involved in

the malignant transformation of hepatic progenitor cells.

Furthermore, we showed that HBx-expressing of HP14.5

cells led to a increased expression of b-catenin and accu-

mulated in cytoplasm, which tended to transfer into cell

nucleus. Over-expression b-catenin in HPC cells also

inhibited the apoptosis of HPCs and blocking b-catenin

expression could neutralize the anti-apoptosis effection of

HBX in HPCs. Therefore, our study demonstrated that the

high expression of HBx protein in HPC cells activated the

Wnt/b-catenin pathway, further supressing the caspase-

dependent apoptosis. This provides a new insight into the

molecular mechanism of HBV-associated HCC.

Acknowledgments We thank Dr. T. -C. He of The University of

Chicago Medical Center for providing the cell lines, vectors and

technical support. The reported work was supported by Research

Grants from the Natural Science Foundation of China (Grant#

81071770, TF and Grant# 81201679, J Y H) .

Conflict of interest The authors report no conflicts of interest.

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