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Introduction
Human hepatocellular carcinoma (HCC) is one of the most
frequent malignant cancers. The carcinogenesis of HCC is a
multifactorial event. Chronic hepatitis B virus (HBV) and
hepatitis C virus (HCV) infections are the most frequent
causes of HCC[1]. Approximately 80% of human HCC are
attributable to HBV infection[2]. Chronic HBV carriers are
100-400 times more likely to develop HCC than
non-carriers[3]. HCV is the second most common cause of HCC after
HBV[4]. Currently, HCC represents more than 4% of all cancer cases
worldwide and causes at least 315 000 deaths every
year[5]. Although early HCC can be cured by surgical resection, many
HCC are asymptomatic, so most HCC patients are not
diagnosed in time.
An effective approach to cancer control is
chemo-prevention. It is known that the therapy of both chronic
HBV and HCV generally involves a long-term course. An
anti-hepatitis drug with an inhibiting or a suppressing effect
on the development of hepatocarcinogenesis, besides its
improvement of abnormal liver function, would be of great
clinical value.
Dimethyl dicarboxylate biphenyl (DDB) is a synthetic
analogue of Schizandrin C, which was isolated from Fructus
Schizandrae chinensis[6]. Since 1983, DDB has been widely
used to treat hepatitis B patients in China and is exported to
Korea, Egypt, Vietnam, Indonesia, Pakistan, and Burma for
the treatment of HBV and HCV. The results of the clinical
application indicated that DDB markedly improved impaired
liver functions, such as the elevated serum transaminase,
bilirubin, a-fetal protein, and symptoms of the patients.
Pharmacologically, DDB has a protective action against
experimental liver injury in mice and
rats[7,8,9]. DDB also had anticancer activity and differentiation-inducing effect on
cancer cells[10]. In the present paper, the chemoprevention
effect of DDB on hepatocellular carcinogenesis in
vitro is studied.
Materials and methods
Chemicals DDB with 99% purity was provided by the
Beijing Union Pharmaceutical Plant. As DDB is not
water-soluble, it was dissolved in dimethyl sulfoxide
(Me2SO) for in vitro use. 3-Methylcholanthrene (3MC),
12-O-tetradecanoyl phorbol 13-acetate (TPA), MTT, and Lucifer
yellow CH were obtained from Sigma Chemical Company.
Other chemicals were of analytical grade and purchased from
Beijing Chemical Company.
Cell culture WB-F344 cells were grown in DMEM
(GIBCO) media containing 10% newborn calf serum, 100 kU/L
penicillin, and 100 mg/L streptomycin in a 37 °C humidified
incubator containing 5% CO2 and 95% air, and passaged
using 0.25% trypsin plus 0.02% EDTA treatment. The
culture medium was changed every other day.
MTT assay Cytotoxicity was determined by MTT assay
according to the method of Mosmann[11]. WB-F344 cells
(3×103cells per well) were plated on 96-well plates, and 24 h
later various concentrations of DDB were added
(0.5 µmol/L-100 µmol/L). The cells were incubated at
37 °C in a CO2 incubator for 72 h. The culture supernatant was sucked out and
MTT 0.5 g/L stock solution was added to each well. After
4 h of incubation, Me2SO was added. The optical density of
each well was determined by a microplate reader at a
wavelength of 570 nm. The values of absorbance were expressed
as relative viable cell number.
In vitro transformation of WB-F344 cells
WB-F344 cells were seeded on 25-cm2 tissue culture flasks containing the
complete medium at a density of
4×103cells per flask. The medium was replaced with the complete medium containing
3MC (2 mg/L) or 0.1% Me2SO 24 h after seeding, and the
cells were incubated for another 72 h. After the removal of
the medium, the cells were washed twice with sterile
phosphate-buffered saline (PBS) and incubated in fresh medium
for 4 d. The cells were then incubated with medium
containing 100 µg/L TPA. The TPA-containing medium was changed
every 2-3 d for 14 d. After sucking out the TPA-containing
medium, the cells were washed twice with sterile PBS and
then incubated in fresh medium containing 10% newborn
calf serum. The fresh medium was changed every 2 d until
d 30. DDB was added to the medium from 24 h after cell
seeding until the end of the experiment. At d 30, three of
these flasks from each group were stained with
Wright-Giemsa, and scored for transformed colonies. The
remainders were used for soft-agar assay.
Soft-agar colony formation assay Cells derived from each
group were seeded separately. Agar (0.6%) in the complete
medium was kept at 44 °C and poured into 6-well plates (2 mL
per well) as to form the lower layer. After the agar medium
had set, 1×104cells per well in 2 mL of 0.3% agar (44 °C) were
layered onto the gelled agar as the form of the upper layer.
The cells were incubated in a humidified atmosphere of 95%
air and 5% CO2 at 37 °C. On d 9 and d 18, 1 mL of 0.3% agar
in the complete medium was added. After 28 d, colonies of
more than 20 cells were counted under contra-phase
microscope.
Cell-cell communication assay The scrape loading/dye
transfer (SL/DT) technique was used to detect GJIC
according to the method of E1-Fouly
et al[12]. WB-F344 cells were
pretreated with various concentrations of DDB for 1 h at
24 h prior to the addition of TPA (100 µg/L) for 1 h. The
other cells were pretreated with 4 µmol/L DDB for 24 h, 48 h,
and 72 h before treatment with TPA. Following incubation,
the cells were washed twice with PBS. Then Lucifer yellow
CH (a fluorescent dye permeating gap-junctional channels)
was added and several scrapes were made with a surgical
steel-bladed scalpel at low light intensities. These scrapes
were performed to ensure that the scrape traversed a large
group of confluent cells. After 3 min incubation, the cells
were washed with PBS again. Dye migration was observed
and photographed with an inverted fluorescent microscope
(Olympus, Japan) at ×200 magnification. The number of dyed
cells represents the ability of cells to communicate via GJIC.
GJIC data are reported as a percentage of the corresponding
mean control value. The data are obtained from 3 views per
plate, pooled 4 separate plates for each point.
Statistical analysis Results are expressed as mean±SD.
To compare mean values between 2 groups, the Student’st-test was used. P<0.05 was considered statistically
signifi-cant.
Results
Cytotoxicity of DDB to WB-F344 cells To select the
appropriate doses of DDB for the present study, the
cytotoxicity of DDB to WB-F344 cells was assessed using the
MTT assay. No significant cytotoxic effect on the cells was
observed when the concentrations of DDB were below
4 µmol/L (Table 1). Therefore, 1 µmol/L,
2 µmol/L, and 4 µmol/L of DDB were used in the subsequent experiments.
Effect of DDB on two-stage transformation of WB-F344
cells A two-stage (initiation and promotion) chemical
induction oncogenesis model with WB-F344 cells was established.
The WB-F344 cells became transformed after 3-MC (2 mg/L)
initiation for 72 h and then TPA (100 µg/L) promotion for
14 d. The transformed cells were grown in a disorganized
multilayer instead of in a monolayer (Figure 1). DDB at
concentrations of 1 µmol/L, 2 µmol/L, and 4 µmol/L markedly
inhibited transformation of WB-F344 cells in a dose-
dependent manner. The average number of transformed foci
decreased dramatically by 10.0%, 37.2%, and 47.4%,
respec-tively, after DDB treatment (Table 2).
Effect of DDB on colony of transformed WB-F344 cells
in soft agar To evaluate the tumorigenic potential of the
treated WB-F344 cells, the efficiency of their soft-agar colony
formation was determined. As shown in Table 3, no colony
formed in soft agar in untreated WB-F344 cells, whereas the
cells initiated with 3-MC and promoted with TPA developed
the transformed phenotype of colony formation in soft agar.
A remarkable increase in colony numbers was observed.
The cells treated with 2 µmol/L and 4 µmol/L DDB
also developed the transformed phenotype of colony formation, but
the colony numbers significantly decreased compared with
the model group.
Effect of DDB on GJIC The GJIC of normal WB-F344
cells was well- characterized and did not decrease during the
experimental incubation period (Figure 2Aa). After
exposing the cells to TPA (100 µg/L) for
1 h, over 85% inhibition of GJIC was detected. The Lucifer yellow CH only stayed at
the incision sites or artificially damaged cells (Figure 2Ab).
When the cells were pretreated with DDB 1 µmol/L,
2 µmol/L, and 4 µmol/L, respectively, for 24 h, a dose-dependent
inhibition of TPA-induced downregulation of GJIC was observed.
The GJIC recovered to 25.6%, 34.6%, and 44.9% of the
control group, respectively (Figure 2B). The time-dependent
inhibitory effect of 4 µmol/L DDB on TPA-induced
down-regulation of GJIC is shown in Figure 3. By the addition of
4 µmol/L DDB for 24 h, 48 h, and 72 h, TPA-induced
downregulation of GJIC was markedly reversed in a
time-dependent manner.
Discussion
WB-F344 cells have often been used in the study of
hepatocarcinogenesis[13]. In the present study, we found
that the anti-hepatitis drug DDB at non-toxic doses
markedly prevented the transformation of WB-F344 cells induced
by 3-MC and TPA in vitro, which expressed as significant
decrease of the number of transformed foci and the
malignant degree of transformed cells.
It is well known that carcinogenesis is a multistage and
multimechanism process, involving the irreversible alteration
of a stem cell (the initiation phase), followed by the clonal
proliferation of the initiated stem cell (the promotion phase),
from which the acquisition of the invasive and metastasis
phenotypes are generated (the progression phase).
Intervention to prevent cancer can occur at each step. For
chemoprevention of carcinogenesis, the development of
anti-tumor promoting agent has been regarded as the most
effective pathway.
Intercellular communication is necessary in multicellular
organisms to maintain tissue homeostasis and to control cell
growth and differentiation. Gap junction channels play an
important role in intercellular communication by providing a
direct pathway for the movement of molecular information,
including ions, polarized and non-polarized molecules up to
a molecular mass of 1 kDa between adjacent
cells[14,15]. Much evidence has been documented to support the hy
pothesis that the downregulation of GJIC is a cellular event
underlying the tumor promotion process, and that any
treatment to prevent downregulation of GJIC is important in
prevention of tumor promotion[16,17]. Many tumor promoters
have been shown to inhibit gap junctional communication
in vitro[18,19]. TPA is a well-known classical inhibitor of cell
communication in most cells, including the WB-F344
cell[20]. In the present study, the underlying mechanisms of DDB
against hepatocarcinogenesis were investigated during the
promotional phase using TPA to inhibit GJIC. The WB-F344
cells are known to have high GJIC. The treatment with TPA
significantly inhibited GJIC, as was determined using the
SL/DT assay. The counteracting effect of DDB on GJIC
inhibition caused by TPA suggests that DDB has a
significant action in maintaining GJIC function, and that it might be
beneficial in preventing tumor promotion.
In summary, the results of the present study suggest
that DDB can prevent the malignant transforming of
WB-F344 cells induced by 3-MC and TPA in
vitro. The restoration of GJIC in the promotion phase should contribute, at
least in part, to the anti-hepatocarcinogenic property of DDB.
We conducted other experiments and found that DDB
significantly inhibited liver carcinogenesis induced by DEN/PB
in mice; the data from these experiments will be published in
another paper soon. Both in vitro and in
vivo experiments demonstrated that DDB had a chemopreventive effect on
hepatocarcinogenesis. It is worthy to pay attention to
whether DDB potentially prevents liver carcinogenesis in
patients with chronic viral hepatitis.
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