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Introduction
Hepatocellular carcinoma (HCC) is a major contributor to
cancer incidence and mortality in the world. The incidence
of HCC is rising worldwide and 80% of the burden is borne
by countries in Asia and sub-Saharan
Africa[1]. Despite
recent advances in diagnostic modalities for HCC, the
disease often develops to an advanced stage before it is
detected clinically, and 5 year survival is less than
10%[2,3]. No effective treatment is currently available. Therefore, there
is a critical need to develop more effective strategies for the
chemotherapy of hepatoma.
Chemotherapy is one of the commonly used strategies in
HCC treatment, especially for unresectable patients.
Conventional chemotherapeutic drugs such as cisplatin (CDDP),
adriamycin, and 5-fluorouracil (5-FU) often have severe side
effects that limit their efficacy. Combination therapy with
multiple drugs or modalities is a common practice in the
treatment of cancers, which can achieve therapeutic effects greater
than those provided by a single drug or modality, and can
reduce the side effects and resistance to drugs.
Chinese herbal medicines are now attracting great
attention in the world. They have also shown promising effects
when combined with chemotherapy and may benefit
patients with HCC[4]. Paeonol (Pae), a major active component
extracted from the herb Pycnostelma
paniculatum (Bunge) K Schum, and the root cortex of Paeonia suffruticosa
And-rews[5], possesses extensive pharmacological activities such
as sedation, hypnosis, antipyresis, analgesic, anti-oxidation,
anti-inflammation, and
immunoregulation[6]. It is a white needle crystal with a relatively low-melting point of 51_52 °C
and has a minimal systemic toxicity
(LD50 3430 mg/kg) when orally administered to
mice[7]. In our previous study, the antineoplastic activity of Pae has been demonstrated both
in cell lines, such as the human erythromyeloid cell line K562,
the breast cancer cell line T6-17, the human hepatoma cell
line Bel-7404, and cervical cancer cell line
HeLa[8], and in animal models bearing HepA
hepatocarcinoma[9,10]. A recent study showed that Pae in low concentration had synergetic
effect with 5-FU, mitomycin C, and CDDP in inhibiting the
proliferation of human colorectal cancer cell line
HT-29[11].
The present study was designed to investigate the
growth-inhibitory and apoptosis-inducing effect of Pae alone
or combined with CDDP in order to develop an effective
combination therapy for HCC.
Materials and methods
Cell culture Human hepatoma cells
HepG2 and SMMC-7721 were purchased from Shanghai Institute of
Hepatocar-cinoma (Shanghai, China), and cultured in Dulbecco's
modified Eagle's medium (DMEM) and RPMI-1640 medium,
respectively. Each was supplemented with 10% fetal bovine
serum (FBS) and incubated at 37 °C in a humid atmosphere
with 5% CO2.
Drugs and chemicals The Pae injection was purchased
from First Pharmaceutical Factory of Shanghai (Shanghai,
China, Cat No 990402, 5 mg/mL); the CDDP injection was
purchased from Nanjing Pharmaceutical Factory (Nanjing,
China, Cat No 20050602, 1 mg/1 mL); DMEM and RPMI-1640
medium were from Gibco BRL Life Technologies (Grand
Island, NY, USA);
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and acridine orange (AO) were
from Sigma (St Louis, MO, USA); the DNA-Prep-Reagents
Kit was purchased from Beckman Coulter (Miami, FL, USA,
Cat No 760279K); rabbit polyclonal antibodies against
human Bcl-2 and Bax were all purchased from Lab Vision
Corporation (Fremont, CA, USA), and the
streptavidin-biotin-peroxidase (S-P) reagents kit was obtained from Fuzhou
Maxim Biotech (Fuzhou, Fujian, China).
In vitro cytotoxicity assay
HepG2 and SMMC-7721 cells were seeded in 96-well plates at a density of
1×103_5×103 cells/well in 100 µL medium overnight. Then the cells were
treated with various concentrations of Pae or CDDP alone
or in combination. After drug exposure for 44 h, the MTT
solution (5 g/L) was added to the plates. The cells were
incubated at 37 °C for another 4 h. The formazan was
dissolved in 150 µL/well DMSO, and the absorbance was
detected at 490 nm using the ELx800 Strip Reader (Bio-Tek,
Winooski, VT, USA). All MTT experiments were performed
in triplicate and repeated at least 3 times. The percentage
of cytotoxicity was calculated as follows: cytotoxicity
(%)=(1_OD490 of experimental
well)/OD490 of control well. The
IC50 (defined as the drug concentration with which 50% cell
growth was inhibited) was assessed from the dose-response
curves.
Analysis of in vitro drug interaction
The coefficient of drug interaction (CDI) was used to analyze the
synergistically inhibitory effect of the drug
combination[12]. CDI was calculated as follows: CDI=AB/(A×B). AB is the ratio of the
2-drug combination group to the control group in
OD490, and A or B is the ratio of the single drug group to the
control group in OD490. Therefore, CDI <1 indicates synergism,
CDI <0.7 indicates a significantly synergistic effect, CDI
=1 indicates additivity, and CDI >1 indicates antagonism.
AO fluorescence staining The cells were cultured
overnight in 6-well plates containing cover slips. After treatment
for 24 h, the cover slips were washed twice with PBS and
fixed with 95% ethanol for 15 min. After being acidified with
1% acetic acid for 30 s, the cover slips were dyed with 0.1 g/L
AO for 10 min. Then the slips were differentiated with 0.1
mol/L CaCl2 for 2 min and washed with PBS 3 times. Finally,
the cover slips were sealed and observed under fluorescence
microscope (Olympus, Shinjuku-ku, Tokyo, Japan).
Flow cytometry assay The cells were cultured in 6-well
plates and allowed to grow to 75%_80% confluency, in
triplicate. Non-adhered cells were removed by gentle
washing, and the medium was removed and replaced with
fresh medium containing Pae and/or CDDP at the desired
concentrations. After exposure to drugs for 24 h, the cells
were collected and centrifuged at 1500 r/min in a 15 mL
tube for 10 min. The cells were washed twice with PBS
and resuspended in 50 µL DNA- Prep LPR (Lyse) at room
temperature for 20 s. After that, 500 µL DNA-Prep Stain
(propidium iodide+RNAse) was added and incubated in
darkness at room temperature for 30 min (according to the
procedure program of DNA-Prep Coulter Reagents Kit).
A minimum of 1×106 cells treated for each group were
analyzed using an EPICS XL-MCL model Coulter counter
(Beckman Coulter, Fullerton, CA, USA). Cell cycle
distribution was analyzed using MacCycle software (Beckman
Coulter, Fullerton, CA, USA).
Immunohistochemical analysis for Bcl-2 and Bax
The cells were cultured overnight in 6-well plates
containing cover slips. After incubation with various
concentrations of Pae or CDDP alone or in combination for 24 h, the
cover slips were washed twice with PBS and fixed in 4%
paraformaldehyde for 25 min. Immunohistochemical
staining for Bcl-2 and Bax was performed according to the
standard S-P method described in the procedure program of
the S-P Reagents Kit (Maxim, Fuzhou, Fujian, China). PBS
10 mmol/L was used as a negative control to replace the
primary antibody.
Analysis of immunohistochemical results The
immunohistochemical results were quantitatively analyzed by
the Biological Image Analysis System (Yokohama, Kanagawa, Japan) which consisted of a Nikon ECLIPSE 80i
biology microscope, Nikon Digital Camera DXM 1200F,
and ACT-1 version 2.63 software (Yokohama, Kanagawa,
Japan), and JEOA 801D Morphologic Biological Image
Analysis software, version 6.0 (Jie Da Technologies,
Nan-jing, Jiangsu, China). The sample was observed on 6
randomly-selected optical fields by microscopy (×400), and an
average A value was measured.
Date analysis Biostatistical analyses were done using
SPSS 11.5 software package (SPSS, Chicago, Illinois, USA).
The results of representative experiments are given as
mean±SD, and mean±SEM for multiple experiments. The
non-parametric Kruskal-Wallis test was used to detect differences
among the different experimental groups. The
Mann-Whitney U-test was subsequently used for statistical
evaluation in 2-group comparisons. Pearson correlation
coefficient was used to analyze continuous independent and
dependent variables. A level of P<0.05 was
accepted as statistically significant.
Results
Inhibitory effect of Pae and CDDP on hepatoma cell
proliferation HepG2 and SMMC-7721 cells incubated with
various concentrations of Pae or CDDP alone for 48 h showed a
dose-dependent reduction of cell viability (Figure 1). The
r values of the dose-effect curves for single-agent Pae on both
HepG2 and SMMC-7721 cell lines were 0.959 and 0.984
(P<
0.01), respectively. Similarly, the r values for CDDP were
0.924 and 0.949 (P<0.01), respectively. However, the
sensitivity of the cells to Pae and CDDP was considerably different.
The cells were more sensitive to CDDP than to Pae, when
comparing the IC50 of CDDP to that of Pae. The
IC50 of Pae on HepG2 and SMMC-7721 cells was [104.77±7.26, 95%
confident limits (CI): 86.74_122.80 and (128.47±9.29) (95% CI:
113.69_143.25 mg/L], respectively (Figure 1A), which was
much more than that of CDDP (0.584±0.060, 95% CI: 0.443_
0.726 mg/L) and 2.889±0.204 (95% CI: 2.380_3.398 mg/L) as
shown in Figure 1B.
Synergistic cytotoxicity of Pae combined with CDDP
To investigate the synergistic inhibitory effects of Pae and
CDDP, 3 doses of Pae (15.63, 31.25, and 62.5 mg/L) were used
in combination with different concentrations of CDDP mixed
at a fixed ratio (1:1, v/v). The results showed that Pae
increas-ed the cytotoxicity of CDDP on
HepG2 and SMMC-7721 cells. For example, in the presence of 15.63, 31.25, and 62.5 mg/L
Pae, the IC50 of CDDP reduced from 0.584±0.060 mg/L to
0.366±0.011, 0.161±0.018, and 0.007±0.002 mg/L,
respectively for HepG2 cells (P<0.01, Figure 2A).
CDI was used to evaluate the nature of the interaction.
There are 2 situations (CDI < or =0.7) shown in Figure 2A
under which the combination makes sense in
HepG2 cells. One is the combination of 0.31 mg/L CDDP with 62.5 mg/L
Pae; the other is the combination of 1.25 mg/L CDDP with
15.63 mg/L Pae. Unexpectedly, the synergistic inhibitory
effects of Pae and CDDP on SMMC-7721 cells required a
large amount of those two agents (Figure 2B) which had the
strongest synergism when 62.5 mg/L Pae was combined with
2.5 mg/L CDDP. This suggests that the synergistic
inhibitory effects of Pae and CDDP depend on cell lines.
Cell apoptosis induced by Pae and CDDP
We then examined whether the synergistic effect of Pae combined with
CDDP also applied to the induction of apoptosis. All cells
incubated with AO had green nuclei and yellow chromatin.
Both of the 2 cell lines treated with Pae and CDDP showed
typically apoptotic changes, such as chromatin
condensation and deformed and fragmented nuclei, especially in the
combination groups (Figures 3A, 4A). The ratio of apoptosis
of 500 cells was calculated. In HepG2 cells (Figure 3B), when
Pae was employed at 62.5 mg/L, the number of apoptotic
cells was only slightly above that of the control. However,
the apoptotic rate rose greatly when treated in combination
with 2.5 mg/L CDDP (Figure 3B). Similar results were found
in SMMC-7721 cells (Figure 4B).
The induction of apoptosis by the treatment groups was
also evident from the FCM(Flow cytometry) assay (Figures
3C, 4C). The sub-G1 peak, which appeared before the
G1 phase that represents apoptotic cell population, was
observed clearly in the 2 cell lines treated with CDDP alone.
The apoptotic peak was dramatically increased when the cells
were exposed to Pae combined with CDDP.
Cell cycle perturbation caused by Pae and CDDP
Mcycle software was used to analyze the kinetic changes of cell
cycle distribution. The HepG2 cells exposed to Pae (31.25
mg/L) or CDDP (1.25 mg/L) alone appeared to move out of
the G0/G1 phase and into the S phase, where the S-phase
fraction increased while the G0/G1
fraction decreased. When treated with the combination of the 2 agents, the
G0/G1 fraction of
HepG2 cells decreased from 71.79%±0.76% to
49.59%±0.89%, and the total S-phase fraction increased from
21.04%±0.58% to 47.07%±1.39% (Table 1). The phenomenon
indicates that the combination of the 2 agents may arrest the
cell cycle at the S phase, which may prevent cells from
entering the M phase. The SMMC-7721 cells exposed to the
combination group could also arrest cells in the S phase.
Effect of Pae and CDDP on the expression of Bcl-2 and
Bax The S-P method was used to examine the expression of
Bcl-2 and Bax. The standard positive Bcl-2 and Bax
expressions were stained brown or yellow mainly in the cytoplasm
or membrane. Bcl-2 and Bax were both expressed in the 2 cell
lines. The expression of Bcl-2 decreased in the treatment
groups, especially in the combination group (Figures
5A,6A). In contrast, there was a significant increase of Bax
expression in the combination group compared to the
control (Figures 5B, 6B). The results were quantitatively
analyzed by the Biological Image Analysis System. The
expression of Bcl-2 was downregulated and that of Bax was
upregulated by Pae and/or CDDP. Correspondingly, the
ratio of Bcl-2/Bax decreased, especially in the combination
group (Figures 5C, 6C).
Discussion
The findings of the present study demonstrated that Pae
and CDDP, used as a single agent, possessed growth
inhibition to HepG2 and SMMC-7721 cells in a dose-dependent
manner. There was a synergistic interaction between Pae
and CDDP in the 2 cell lines. The cytotoxity of the
combination group was significantly higher than that treated with
Pae or CDDP alone in appropriate concentrations. We also
found that the interaction between Pae and CDDP was
specific to each cell line. In HepG2 cells, at lower concentrations
of Pae and CDDP, the combination was synergistic. The
synergistic effect was the most prominent (CDI <0.7) when
15.63 mg/L Pae was combined with 1.25 mg/L CDDP in the
HepG2 cells. Treatment with a combination of
chemotherapeutic agents resulted in an improved response as well as
the ability to use less toxic concentrations of the drugs, which
indicates that the combination of CDDP and Pae in certain
concentrations could result in a synergistic effect. Our
results are in accordance with the findings of Ji
et al[11] who demonstrated that Pae in low concentrations had a
synergetic effect with 5-FU, MMC, and CDDP in inhibiting the
proliferation of the human colorectal cancer cell line HT-29.
Similar results were observed in the SMMC-7721 cells, but
with different sensitivity. As many reports have shown,
different cell lines are different in their susceptibility to
drugs[13_16]. In this study, the 2 cell lines are both adherent,
epithelial-like cells, but from different origins. The
HepG2 cell was established from the tumor tissue of a 15-year-old
Argentine boy with hepatocellular carcinoma in 1975, which
was reported to produce a variety of proteins such as
alpha-fetoprotein, albumin, alpha2-macroglobulin,
alpha1-antitrypsin, and transferrin. The SMMC-7721 cell was
established from the tumor tissue of a 56-year-old Chinese
man with HCC in 1980. The different origins and different
biological activities may be one of the reasons for the
different susceptibility to the drugs.
Although the exact mechanism of the cytotoxicity of Pae
against tumor cells is not entirely clear, many potential
mechanisms have been proposed for the growth inhibition by Pae
on cultured cells and animal models. These mechanisms
include the induction of
apoptosis[17,18] and immuno-regulation, such as promoted lymphocyte proliferation,
interleukin-2 production by splenocytes, and TNF-α
production by PMf (peritoneal macrophages) from model
mice[9,10]. Apoptosis is a mechanism by which cells undergo
death to control cell proliferation or in response to DNA
damage. The hypothesis that failure to undergo apoptosis
contributes to the development of resistance to anticancer
agents has been the subject of extensive
research[19,20]. Therefore, agents that facilitate apoptosis should improve
therapeutic efficacy. Previous studies have demonstrated
that Pae could induce apoptosis in
K562[17] and HT-29
cells[18]. To investigate the apoptosis-inducing effect of Pae
as a single agent and combined with CDDP in hepatoma
cells, the morphological changes and apoptotic rate were
detected. The cells treated with the drugs showed the
typical characteristics of apoptosis, which were more prominent
in the combination group. Similarly, an apoptotic peak
appeared before the G1 phase when treated with Pae or CDDP
alone, and a significant synergistic effect on the induction
of apoptosis was observed in the combination group.
We also found that the HepG2 and SMMC-7721 cells
exposed to Pae alone for 24 h showed depletion of the
G1 fraction and accumulation in the S phase. Accumulation in
the S phase has also been reported by Liu et
al[18], in which Pae could induce cell cycle perturbation and HT-29 cells
attracted in the S phase increased, while cells of the
G0/G1 and G2/M phases decreased. The cytotoxic effect of CDDP
is generally considered to be non-cell-cycle
specific[21]. CDDP can cause perturbations in cell cycle
distribution[22] and it is most specific to
G1-phase cells, but also has strong effect on cells in the S
phase[23]. Our data also suggested that the combination group exhibited enhanced S-phase
arrest, along with depletion of the
G0/G1 fraction, which may be one of the mechanisms related to these interactions.
To examine the mechanism of apoptosis, we examined
the expression of the Bcl-2 protein family, which is an
important regulator of apoptosis[24]. The Bcl-2 family includes
pro-apoptotic members such as Bax, Bak, Bad, Bcl-Xs, Bid,
Bik, Bim, and Hrk, and anti-apoptotic members such Bcl-2,
Bcl-XL, Bcl-W, Bfl-1, and
Mcl-1[25]. These effects are more dependent on the balance between Bcl-2 and Bax than on
the Bcl-2 quantity alone[26_28]. In the present study,
treatment with Pae and/or CDDP decreased the expression of
Bcl-2 and increased the expression of Bax, especially in
the combination group. Furthermore, a significant decrease
in the ratio of Bcl-2/Bax was observed when Pae was treated
in combination with CDDP, which correlated with the
incidence of apoptosis. One possible explanation for the
synergistic interaction could be suggested. The
upregula-tion or downregulation of the Bcl-2 protein family by Pae
and/or CDDP might be the mechanism to introduce apoptosis.
In summary, the results obtained in the present study
indicate that Pae in combination with CDDP has significantly
synergistic growth-inhibitory and apoptosis-inducing effect
on the human hepatoma cell lines HepG2 and SMMC-7721,
which may be related with cell cycle arrest and the
upre-gulation of the Bcl-2 family. Pae is expected to be effective
and useful as a new agent in HCC treatment in the future.
Acknowledgement
We thank Dr Zhi-min ZHAI and Ms Qing LI (Central
Laboratory of the Provincial Hospital of Anhui) for the FACS
analysis.
References
1 McGlynn KA, London WT. Epidemiology and natural history
of hepatocellular carcinoma. Best Pract Res Clin Gastroenterol
2005; 19: 3_23.
2 Teo EK, Fock KM. Hepatocellular carcinoma: an Asian
perspective. Dig Dis 2001; 19: 263_8.
3 Johnson PJ. Hepatocellular carcinoma: is current therapy really
altering outcome? Gut 2002; 51: 459_62.
4 Shu X, McCulloch M, Xiao H, Broffman M, Gao J. Chinese
herbal medicine and chemotherapy in the treatment of
hepatocellular carcinoma: a meta-analysis of randomized controlled
trials. Integr Cancer Ther 2005; 4: 219_29.
5 Riley CM, Ren TC. Simple method for the determination of
paeonol in human and rabbit plasma by high performance liquid
chromatography using solid-phase extraction and ultraviolet
detection. J Chromatogr 1989; 489: 432_7.
6 Sun YC, Shen YX, Sun GP. Advances in the studies of major
pharmacological activity of paeonol. Chin Tradit Pat Med 2004;
26: 579_82. Chinese.
7 Jiang SP, Chen YX. Advances and application in the studies of
the Pycnostelma paniculatum (Bunge) K.S. and paeonol. China
J Chin Mater Med 1994; 19: 311_4. Chinese.
8 Sun GP, Wang H, Shen YX, Xu SY. Inhibitory effects of paeonol
on the proliferation on four tumor cell lines. Anhui Med Pharm
J 2004; 8: 85_7. Chinese.
9 Sun GP, Shen YX, Zhang LL, Zhou AW, Wei W, Xu SY. Study on
immunomodulation and antitumor activity of paeonol in HepA
tumor mice. Chin Pharm Bull 2003; 19: 160_2. Chinese.
10 Sun GP, Shen YX, Zhang LL, Wang H, Wei W, Xu SY.
Anti-tumor effect of paeonol in vitro and
in vivo. Acta Univ Med Anhui 2002; 37: 183_5. Chinese.
11 Ji CY, Tan SY, Liu CQ. Inhibitory effect of paeonol on the
proliferation of human colorectal cancer cell line HT-29 and its
synergistic effect with chemotherapy agents. Chin J Clin
Gastro-enterol 2005; 17: 122_4. Chinese.
12 Cao SS, Zhen YS. Potentiation of antimetabolite antitumor
activity in vivo by dipyridamole and amphotericin B. Cancer
Chemother Pharmacol 1989; 24: 181_6.
13 Horie N, Hirabayashi N, Takahashi Y, Miyauchi Y, Taguchi H,
Takeishi K. Synergistic effect of green tea catechins on cell
growth and apoptosis induction in gastric carcinoma cells. Biol
Pharm Bull 2005; 28: 574_9.
14 Qi YL, Liao F, Zhao CQ, Lin YD, Zuo MX. Cytotoxicity,
apoptosis induction, and mitotic arrest by a novel
podophyllotoxin glucoside, 4DPG, in tumor cells. Acta Pharmacol Sin 2005;
26: 1000_8.
15 Tyagi AK, Agarwal C, Chan DC, Agarwal R. Synergistic
anti-cancer effects of silibinin with conventional cytotoxic agents
doxorubicin, cisplatin and carboplatin against human breast
carcinoma MCF-7 and MDA-MB468 cells. Oncol Rep 2004; 11:
493_9.
16 Yim D, Singh RP, Agarwal C, Lee S, Chi H, Agarwal R. A novel
anticancer agent, decursin, induces G1 arrest and apoptosis in
human prostate carcinoma cells. Cancer Res 2005; 65: 1035_44.
17 Sun GP, Wang H, Shen YX, Zhai ZM, Wei W, Xu SY. Study on
effects of paeonol in inhibiting growth of K562 and inducing its
apoptosis. Chin Pharm Bull 2004; 20: 550_2. Chinese.
18 Liu CQ, Tan SY, Ji CY, Luo HS, Yu JP. The effects of paeonol on
inhibiting the proliferation of human colorectal cancer cell line
HT-29 and its molecule mechanism. Chin Pharm Bull 2005; 21:
1251_4. Chinese.
19 Gottesman MM. Mechanisms of cancer drug resistance. Annu
Rev Med 2002; 53: 615_27.
20 Kaufmann SH, Vaux DL. Alterations in the apoptotic machinery
and their potential role in anticancer drug resistance. Oncogene
2003; 22: 7414_30.
21 Schweitzer VG. Cisplatin-induced cytotoxicity: the effect of
pigmentation and inhibitory agents. Laryngoscope 1993; 103:
1_52.
22 Nguyen HN, Sevin BU, Averette HE, Perras J, Ramos R, Donato
D, et al. Cell cycle perturbations of platinum derivatives on two
ovarian cancer cell lines. Cancer Invest 1993; 11: 264_75.
23 Potter AJ, Rabinovitch PS. The cell cycle phases of DNA
damage and repair initiated by topoisomerase II-targeting
chemotherapeutic drugs. Mutat Res 2005; 572: 27_44.
24 Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell
survival. Science 1998; 281: 1322_6.
25 Reed JC. Bcl-2 and the regulation of programmed cell death. J
Cell Biol 1994; 124: 1_6.
26 Reed JC. Bcl-2 family proteins: regulators of apoptosis and
chemoresistance in hematologic malignancies. Semin Hematol
1997; 34: 9_19.
27 Zhang CL, Wu LJ, Tashiro S, Onodera S, Ikejima T. Oridonin
induces apoptosis of HeLa cells via altering expression of
Bcl-2/Bax and activating caspase-3/ICAD pathway. Acta Pharmacol
Sin 2004; 25: 691_8.
28 Zhong L, Li CM, Hao XJ, Lou LG. Induction of leukemia cell
apoptosis by cheliensisin A involves down-regulation of Bcl-2
expression. Acta Pharmacol Sin 2005; 26: 623_8. |
