Synergistic effect of 5-fluorouracil and the flavanoid oroxylin A on HepG2 human hepatocellular carcinoma and on H22 transplanted mice.

Abstract

To investigate the synergistic inhibitory effects of the combination of 5-fluorouracil (5-FU) with the natural flavanoid oroxylin A on human hepatocellular carcinoma cells HepG2 in vitro and on transplanted murine hepatoma 22 (H22) tumors in vivo and the preliminary mechanisms.
The inhibitory effects of 5-FU combined with the natural flavanoid oroxylin A in vitro were detected by MTT assay and the effects in vivo were investigated by transplanted H22 mice model. DAPI staining and Annexin V/propidium iodide (PI) double staining were used to detect the cell morphological changes and apoptosis. The mRNA levels of thymidine synthetase (TS) and dihydropyrimidine dehydrogenase (DPD) in HepG2 cells after oroxylin A and 5-FU combination treatment were observed by quantitative real-time PCR. Western blotting assay was used to reveal the expressions of apoptotic-inducing proteins P53, cleaved PARP, COX-2, Bcl-2, and pro-caspase3.
Oroxylin A in combination with 5-FU presented synergistic effect (CI<1) on HepG2 cells in vitro when the inhibitory rate was higher than 7.5%. The inhibitory rate on H22 murine solid tumor in vivo in the combination group was higher than monotherapy. 5-FU combined with oroxylin A exerted stronger apoptotic induction in HepG2 cells than either single drug treatment. Quantitative real-time PCR discovered the downregulation of TS mRNA and DPD mRNA in HepG2 cells after combination treatment. Western blotting assay revealed oroxylin A enhanced 5-FU-induced apoptosis in HepG2 cells by elevating the expressions of apoptotic-inducing proteins P53 and cleaved PARP and decreasing the expression of apoptotic-inhibitory proteins COX-2, Bcl-2, and pro-caspase3.
The anti-hepatocellular carcinoma effects in vitro and in vivo of 5-FU and oroxylin A combinations were synergistic and oroxylin A increased the sensitivity of HepG2 cells to 5-FU by modulating the metabolic enzymes of 5-FU and apoptotic-related proteins.

Full text

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ORIGINAL ARTICLE
Synergistic effect of 5-fluorouracil and the flavanoid oroxylin A
on HepG2 human hepatocellular carcinoma
and on H22transplanted mice
Li Zhao Æ Æ Zhen Chen Æ Æ Jun Wang Æ Æ Li Yang Æ Æ
Qing Zhao Æ Æ Jia Wang Æ Æ Qi Qi Æ Æ Rong Mu Æ Æ
Qi-Dong You Æ Æ Qing-Long Guo
Received: 28 April 2009/Accepted: 12 June 2009/Published online: 8 July 2009
? Springer-Verlag 2009
Abstract
Aim
the combination of 5-fluorouracil (5-FU) with the natural
flavanoid oroxylin A on human hepatocellular carcinoma
cells HepG2 in vitro and on transplanted murine hepatoma
22 (H22) tumors in vivo and the preliminary mechanisms.
Methods
The inhibitory effects of 5-FU combined with
the natural flavanoid oroxylin A in vitro were detected by
MTT assay and the effects in vivo were investigated by
transplanted H22mice model. DAPI staining and Annexin
V/propidium iodide (PI) double staining were used to
detect the cell morphological changes and apoptosis. The
mRNA levels of thymidine synthetase (TS) and dihydro-
pyrimidine dehydrogenase (DPD) in HepG2 cells after
oroxylin A and 5-FU combination treatment were observed
by quantitative real-time PCR. Western blotting assay was
used to reveal the expressions of apoptotic-inducing pro-
teins P53, cleaved PARP, COX-2, Bcl-2, and pro-caspase3.
To investigate the synergistic inhibitory effects of
Results
synergistic effect (CI\1) on HepG2 cells in vitro when
the inhibitory rate was higher than 7.5%. The inhibitory
rate on H22murine solid tumor in vivo in the combination
group was higher than monotherapy. 5-FU combined with
oroxylin A exerted stronger apoptotic induction in HepG2
cells than either single drug treatment. Quantitative real-
time PCR discovered the downregulation of TS mRNA
and DPD mRNA in HepG2 cells after combination
treatment. Western blotting assay revealed oroxylin A
enhanced 5-FU-induced apoptosis in HepG2 cells by
elevating the expressions of apoptotic-inducing proteins
P53 and cleaved PARP and decreasing the expression of
apoptotic-inhibitory proteins COX-2, Bcl-2, and pro-
caspase3.
Conclusion
The anti-hepatocellular carcinoma effects in
vitro and in vivo of 5-FU and oroxylin A combinations
were synergistic and oroxylin A increased the sensitivity of
HepG2 cells to 5-FU by modulating the metabolic enzymes
of 5-FU and apoptotic-related proteins.
Oroxylin A in combination with 5-FU presented
Keywords
Hepatocellular carcinoma ? Synergistic effect
5-Fluorouracil ? Oroxylin A ?
Introduction
Hepatocellular carcinoma (HCC) is the fifth most common
malignancy in the world. Although surgical managements
ornon-surgicaltherapeutic
employed, HCC is rarely curative. Chemotherapy is used in
a variety of cancer treatments. Single-agent responses are
usually partial and relatively short. Moreover, the toxicity
of the single agent to normal tissues has been one of the
major obstacles to successful cancer treatment. Therefore,
modalitieshavebeen
Li Zhao, Zhen Chen contributed equally to this article.
L. Zhao ? J. Wang ? L. Yang ? Q. Zhao ? J. Wang ? Q. Qi ?
R. Mu ? Q.-L. Guo (&)
Jiangsu Key Laboratory of Carcinogenesis and Intervention,
China Pharmaceutical University, 24 Tongjiaxiang,
210009 Nanjing, China
e-mail: anticancer_drug@yahoo.com.cn
Q.-D. You (&)
Department of Medicinal Chemistry, China Pharmaceutical
University, 24 Tongjiaxiang, 210009 Nanjing, China
e-mail: youqidong@gmail.com
Z. Chen
Department of Pharmacology, China Pharmaceutical University,
24 Tongjiaxiang, 210009 Nanjing, China
123
Cancer Chemother Pharmacol (2010) 65:481–489
DOI 10.1007/s00280-009-1053-2

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combined treatments with several chemotherapy drugs are
often used to enhance the therapeutic effect.
Oroxylin A (Fig. 1), a bioactive flavonoid, is extracted
from the root of a traditional Chinese medicine—Scutel-
laria baicalensis Georgi [16]. It has been shown to be a
promising candidate for selective and effective manage-
ment of inflammation [22]. Chen [2] reported that oroxylin
A inhibited lipopolysaccharide (LPS)-induced expression
of nitric oxide synthase (iNOS) and cyclooxygenase-2
(COX-2) in a concentration-dependent manner. Huang [10]
also reported oroxylin A exhibited significant antioxidative
and free-radical scavenging activities. The effects of or-
oxylin A reversing scopolamine and Abeta (25–35) pep-
tide-induced memory and congnitire impairment via the
GABAergic neurotransmitter system were reported by Kim
[12, 13]. Besides, oroxylin A could inhibit diclofenac 4-
hydroxylation (CYP2C9) activity in human liver micro-
somes to protect liver [14] and be a potent anti-respiratory
syncytial virus component [18]. Recently, our studies
found oroxylin A induced human hepatocellular carcinoma
cell line HepG2 apoptosis by modulating the concerted
expression of Bcl-2, Bax, and pro-caspase-3 proteins [9]
and inhibited the growth of human gastric carcinoma BGC-
823 cells by inducing G2/M phase cell-cycle arrest via
inhibiting Cdk7-mediated expression of Cdc2/p34 [30].
The antimetabolite 5-fluorouracil (5-FU) is widely used
in many types of tumors for over 20 years, but responses are
obtained in only 10–30% of patients with advanced disease.
It exerts antitumor effects by disturbing RNA processing
and inhibiting DNA synthesis. It may be converted to 5-
fluorouridine-50-monophosphate (FUMP) either directly by
orotate phosphoribosyltransferase or by uridine phosphor-
ylase and uridine kinase. FUMP may then be converted to5-
fluorouridine-50-triphosphate (FUTP) which, after incorpo-
ration into RNA, can interfere with the synthesis and
function of all classes of RNA. FUMP may be converted
[after reduction of 5-fluorouridine-50-diphosphate (FUDP)
by ribonucleotide reducase] to 5-fluoro-20-deoxyuridine-50-
monophosphate (FdUMP) and hence DNA synthesis. Thus,
activation of 5-FU may give rise to inhibition of DNA and/
or RNA metabolism and DNA synthesis [19, 21].
Thymidine synthetase (TS) is a rate-limiting enzyme in
pyrimidine de novo deoxynucleotide biosynthesis and is
thereforeanexcellenttarget for chemotherapeutic
strategies. TS plays a central role in DNA synthesis in the
reductive methylation of deoxyuridine-50-monophosphate
(dUMP) to deoxythymidine-50-monophosphate (dTMP).
The cosubstrate for TS is the reduced-folate cofactor, 5,10-
methylenetetrahydrofolate (CH2THF), which forms a ter-
nary complex with dUMP and TS. FdUMP, the active
metabolite of 5-FU competes with dUMP, and can form a
ternary complex which does not dissociate readily because
the methyl group cannot be transferred [27]. Cancer cells
with high levels of FdUMP and low levels of TS are thus
known to be sensitive to 5-FU [20]. Dihydropyrimidine
dehydrogenase (DPD) is a rate-limiting enzyme involved
in the degradation of 5-FU and inactivates over 80% of
administered 5-FU in vivo. Inactivation of 5-FU by DPD
seemed to be a mechanism of clinical resistance to 5-FU,
vigorous efforts have been made to design inhibitors of
DPD [20]. The activity of DPD in tumor cells is critical to
therapeutic effects of 5-FU, thus its inhibition improves the
antitumor efficacy and the therapeutic index of 5-FU [8].
In this study, we tested the effects of 5-FU and oroxylin
A alone and in combination regarding their activities
against hepatocellular carcinoma in vitro and in vivo, and
investigated the possible mechanisms involved.
Materials and methods
Chemicals
Oroxylin A was isolated from S. radix according to the
protocols reported previously [16] with slight modifica-
tions. Samples containing 99% or higher oroxylin A were
used in all experiments unless otherwise indicated. Or-
oxylin A was dissolved at a concentration of 100 mM in
100% DMSO (Amresco, Amresco Inc., Solon, Ohio) as a
stock solution, stored at -20?C, and diluted with medium
before each experiment. The final DMSO concentration did
not exceed 0.1% throughout the study. 5-FU (25 mg/ml)
was purchased from Tianjin Amino Acids Company
(Tianjin, China) and diluted to various concentrations with
culture medium. Primary antibodies for pro-caspase3, P53,
and b-actin were obtained from Santa Cruz Biotechnology
(Santa Cruz Biotechnology Inc., CA). Primary antibodies
for Bcl-2 and PARP were purchased from Cell Signaling
Technology (Beverly, MA). Primary antibody for COX-2
was the product of Bioworld Technology Inc. (China).
IRDye
800 conjugated secondary antibodies were
obtained from Rockland Inc. (Bedford, PA).
TM
Cell culture
Human hepatocellular carcinoma cell line HepG2 was
purchased from Cell Bank of Shanghai Institute of
Fig. 1 Molecular structure of oroxylin A. The molecular formula of
oroxylin A is C16H12O5and the molecular weight is 284
482 Cancer Chemother Pharmacol (2010) 65:481–489
123

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Biochemistry & Cell Biology, Chinese Academy of Sci-
ences. The cells were cultured in RPMI-1640 medium
(GIBCO, NY) supplemented with 10% heat-inactivated
fetal bovine serum (Sijiqing, Zhejiang, China), 100 U/ml
benzyl penicillin, and 100 U/ml streptomycin in a humid-
ified environment with 5% CO2at 37?C.
Murine hepatoma 22 (H22) cells, provided by Jiangsu
Institute of Antitumor Pharmaceuticals, were maintained in
RPMI-1640 medium supplemented with 10% heat-inacti-
vated calf serum (Sijiqing, Hangzhou, China), 100 U/ml
penicillin G, and 100 U/ml streptomycin, pH 7.4 in a
water-jacketed CO2incubator (Thermo Forma, USA) with
a humidified atmosphere of 5% CO2at 37?C.
Animals
Male kunming mice with body weight ranging from 18 to
22 g were provided by the Animals Facility at China
Pharmaceutical University [No. SCXK (Su) 2002-0011].
Animals were maintained in a pathogen-free environment
(23 ± 2?C, 55 ± 5% humidity) on a 12-h light/12-h dark
cycle with food and water supplied adlibitum throughout
the experimental period.
Cell growth inhibition assay
HepG2 cells were cultured in the presence of oroxylin A or
5-FU or both drugs for 48 h, the growth inhibitory effect
was determined by measuring MTT (3-[4,5-dimethylthia-
zol-2-yl]-2,5-diphenyltetrazolium bromide) assay. The
inhibitory ratio was calculated by the following formula:
inhibitory ratio (%) = 1 - average absorbance of treated
group/average absorbance of control group 9 100%.
Combined effect evaluation
Drug interaction between oroxylin A and 5-FU was
assessed at a fixed concentration ratio of 1:1. The combi-
nation index (CI) was calculated as follows:
CI ¼
D
DX
ð Þ1
ðÞ1
þ
D
DX
ð Þ2
ðÞ2
where (DX)1and (DX)2are the doses for oroxylin A and 5-
FU in a combination that inhibits 50% cell growth, and
(D)1and (D)2are the doses for each drug alone that inhibit
50% cell growth. CI\1, CI = 1, and CI[1 indicated
synergistic, additive, and antagonistic effects, respectively
[4, 6]. Data analysis was performed by the Calcusyn soft-
ware (Biosoft, Oxford, UK).
DAPI staining
Human hepatocellular carcinoma HepG2 cells were cul-
tured in RPMI-1640 till mid-log phase. Then 50-lM
oroxylin A, 750-lM 5-FU alone and the both were exposed
to cells for 48 h. The morphology of the cells was moni-
tored under an inverted light microscope. All floating and
attached cells were harvested with 0.02% (w/v) EDTA and
0.25% (w/v) trypsinase. The cells were fixed with ice-cold
4% paraform for 20 min and washed with ice-cold PBS,
then permeabilized with 0.3% Triton X-100 and washed
with ice-cold PBS, stained with fluorochrome dye DAPI
(Santa Cruz, USA) and observed under a fluorescence
microscope (OlympusIX51, Japan) with a peak-excitation
wave length of 340 nm.
Annexin V/PI double staining
Cells were incubated for 48 h with either oroxylin A or 5-
FU or in combination. Apoptotic cells were identified by
the Annexin V-FITC Apoptosis Detection kit (Biovision,
Moutain View, CA) according to the manufacturer’s
instructions. Flow cytometric analysis was performed
immediately after supravital staining. Data acquisition and
analysis were performed in a Becton Dickinson FAC-
SCaliburflow cytometer
(Franklin Lakes, NJ, USA).
usingCellQuest software
Quantitative real-time reverse transcription-PCR
analysis
Cells were incubated for 48 h with either oroxylin A or 5-
FU or in combination. Then total cellular RNA was
extracted using the TriPure Solution (Takara, Takara Bio
Inc., Otsu, Shiga, Japan) following the manufacturer’s
instructions. cDNA of 1 lg total RNA was synthesized
using random primers and Primescript reverse transcrip-
tase (Takara, Takara Bio Inc., Otsu, Shiga, Japan).
Quantitative PCRs for indicated genes were carried out
using SYBR green qPCR kit (Takara, Takara Bio Inc.,
Otsu, Shiga, Japan) by a fluorescent temperature cycler
(Real-Time PCR System, Eppendorf, Hamburg, Ger-
many). The primers used in the reactions are as follows:
TS 50-CAC CCT GTC GGT ATT CG-30and 50-CCA
CTG GAA GCC ATA AAC T-30; DPD 50-AAA GAA
GCC TTG AGC T-30and 50-TAG CCA AAC CAA CGA
C-30. Thermocycling was done in a final volume of 20 ll
containing 2-ll cDNA sample (1 lg/ll), 0.4 ll of each
primer (10 nM), 10-ll SYBR green I, 7.2-ll H2O. After
an initial denaturation at 94?C for 10 s, 40 cycles at 94?C
for 30 s, annealing at 55?C for 30 s, and 72?C for 30 s
were carried out. All cDNA samples were synthesized in
parallel, and PCR reactions were run in triplicate. mRNA
levels were derived from standard curves and expressed
as relative changes after normalization versus GAPDH
mRNA levels [28].
Cancer Chemother Pharmacol (2010) 65:481–489483
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Western blot analysis
Cells were treated with oroxylin A or 5-FU single or in
combination. Then they were lysed in lysis buffer
(100 mM Tris–HCl, pH 6.8, 4% (m) SDS, 20% (v) glyc-
erol, 200 mM b-mercaptoethanol, 1 mM PMSF, and 1 g/
ml aprotinin) for 1 h on the ice. The lysates were clarified
by centrifugation (12,000 rpm) at 4?C for 15 min. The
protein concentration in the supernatants was detected by a
Varioskanmultimode microplate
(Thermo, Waltham, MA, USA). Then equal amount of
protein was separated by SDS-PAGE [34]. Proteins were
detected using specific antibodies of Bcl-2, P53, COX-2,
PARP, pro-caspase3, and b-actin followed by IRDye
conjugated secondary antibodies for 1 h at 37?C. Detection
was performed by the Odyssey Infrared Imaging System
(LI-COR Inc., Superior St. Lincoln, NE, USA). All blots
were stripped and probed with polyclonal anti-b-actin
antibody to ascertain equal loading of the proteins.
spectrophotometer
TM800-
Inhibitory effects on H22murine solid tumor
Murine hepatoma 22 (H22) cells were diluted with icy 0.9%
saline and inoculated subcutaneously at right axilla of mice
(5 9 106viable cells/each ml) [29]. Twenty-four hours
after inoculation, mice were divided randomly into four
groups (with 10 mice/group): saline tumor control group;
oroxylin A 1,000 mg/(kg/day) orally group; 5-FU 10 mg/
(kg/day) group; and oroxylin A ? 5-FU combination
group. Oroxylin A was administered orally (po) and 5-FU
was given intravenously (iv). Both were given once every
day. From the third day after treatment, the tumors were
measured continuously. Tumor volume (TV) was calcu-
lated using the following formula:
?
where d and D were the shortest and the longest diameter,
respectively.
At the seventh day after treatment, the mice were killed
and tumors were ablated carefully and weighed after
washing off any remaining blood with PBS. The dose of
oroxylin A was 1,000 mg/(kg/day), which was about one-
fifth of LD50 value. The 5-FU 10 mg/(kg/day) was deter-
mined as a half of its effective dose 20 mg/(kg/day). This
study was approved in SPF Animal Laboratory of China
Pharmaceutical University.
TV mm3
?¼ d2? D=2
Statistical analysis
All results shown represent means ± SEM from triplicate
experiments performed in a parallel manner unless other-
wise indicated. Statistical analyses were performed using
one-way ANOVA by SPSS 11.5 software.
Results
Effects of 5-FU or oroxylin A on the inhibition of cell
viability and combination index of two drugs mixture
The inhibitionary effect of oroxylin A or 5-FU on cell
viability in HepG2 cells was assessed after 48 h exposure,
following a 24 h cultivation in drug-free medium. Results
indicated that oroxylin A and 5-FU showed inhibitory
effect by a concentration-dependent manner. As shown in
Fig. 2a, b, the IC50 of oroxylin A and 5-FU was
39.79 ± 2.84 and 792.61 ± 14.65 lM, respectively. To
detect the inhibitory effect of the combination treatment,
cells were exposed to oroxylin A and 5-FU concurrently
for 48 h at a fixed ratio (oroxylin A IC50:5-FU IC50ratios
were 1:1) [4]. Data showed the CI values were\1 (Fig. 2c)
when the values of fraction affects (Fa) were higher than
0.075 (at the point, the concentration of oroxylin A and 5-
FU was about 0.6 and 1.4 lM, respectively), which indi-
cated that the combination of oroxylin A and 5-FU exerted
a synergistic inhibitory effect on the proliferation of
HepG2 cells at concentrations higher than the threshold
values (drug concentrations when CI = 1). Moreover,
when the value of Fa is 0.5 (the inhibitory rate was 50%),
the C5-FUis 157.57 lM, which is only about 20% of the
IC50value of 5-FU single treatment.
Cell morphological assessment by DAPI staining
After oroxylin A (50 lM) or 5-FU (750 lM) or the com-
bination treatments for 48 h, the morphological changes of
the cells were examined by light microscopy. As shown in
Fig. 3a, untreated HepG2 cells stained with equal intensity
of DAPI and demonstrated homogeneously distributed
chromatin within their nuclei, while treated cells displayed
chromatin condensation and nucleolus condensation (pyk-
nosis), which indicated an early apoptotic event (arrow
designating) [31]. Moreover, we observed that the apop-
tosis events in the combination group were more distin-
guished than that of either single drug treatment group.
These results suggested that oroxylin A increase the cyto-
toxic effect of 5-FU on HepG2 cells.
Apoptosis induced by 5-FU or oroxylin A
or in combination of them by Annexin V/PI staining
Apoptosis induced by oroxylin A and 5-FU in HepG2 cells
was analyzed using Annexin V/PI staining. Compared with
control group, the proportion of apoptotic cells increased
after cells were exposed to 50 lM of oroxylin A or 750 lM
of 5-FU or both drugs for 48 h with the early apoptosis rate
17.5%, 21.0%, and 26.3%, respectively. Among which the
apoptotic induction of the combination was the strongest.
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The condition of the late apoptosis and dead cells was
similar with that of the early apoptotic cells (Fig. 3b).
These results indicated that oroxylin A could increase the
apoptotic-inducing effect and cytotoxic effect of 5-FU in
HepG2 cells.
Changes of the mRNA levels of the TS and DPD
metabolic enzymes after treatment with 5-FU or
oroxylin A or the combination in HepG2 cells
To further elucidate the synergistic mechanism of 5-FU
and oroxylin A, we examined the effect of them on the
mRNA levels of TS and DPD on HepG2 cells. As shown in
Fig. 4, 5-FU treatment alone caused a 5.18 ± 0.39-fold
increase at mRNA level of TS in HepG2 cells compared
with the control group (P\0.01). In contrast, the dual
treatment decreased it nearly to one quarter at 1.43 ± 0.13-
fold of control. And, the DPD mRNA level in 5-FU
treatment group was 9.31 ± 0.17-fold increase compared
with the control group (P\0.01), while the level in
combined group was down-regulated to about one-third of
5-FU group at 2.92 ± 0.15 (compared with control,
P\0.01).
Western blot analysis for apoptosis-related proteins
in HepG2 cells
To further illuminate the mechanism of co-treatment with
oroxylin A which may enhance the apoptotic effect
induced by 5-FU in HepG2 cells, the expressions of some
apoptotic related proteins were investigated. Expressions of
COX-2, Bcl-2, and P53 were detected after cells were
incubated with either oroxylin A or 5-FU or in combination
0
0.00
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0.200.40 0.600.80 1.00
Fraction Effect
Combination Index
CI=1
Fa=0.075
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
0.290.59 0.89 1.191.49 1.802.10 2.402.70
log (dose of Oroxylin-A (µM))
Inhibitory rate
0%
10%
20%
30%
40%
50%
60%
70%
80%
1.301.702.002.28
log (dose of 5-FU (µM))
2.582.883.18 3.483.78
Inhibitory rate
Fig. 2 Inhibitory effect of oroxylin A, 5-FU and the mean-effect of
these two drugs on HepG2 cells. Effects of two single drugs on
HepG2 cells and the combination index of the two drugs. HepG2 cells
were treated with various concentration of oroxylin A (a) or 5-FU (b)
alone for 48 h. Cell viability was determined using MTT assay
Fig. 3 Apoptosis assessment in HepG2 cells. a Nucleolus morpho-
logical changes observed by fluorescence microscope (2009). HepG2
cells were incubated with 50 lM oroxylin A, 750 lM 5-FU alone and
the combination of them for 48 h. DAPI staining was used to
visualize the cell nucleus. The nucleolus changes of HepG2 cells were
observed under the fluorescent microscope. b Apoptosis assessment
by Annexin V/PI staining in HepG2 cells. Apoptosis in HepG2 cells
treated with oroxylin A (50 lM), 5-FU (750 lM) either in combi-
nation or alone for 48 h. Early apoptotic cells were Annexin V-
positive, PI-negative cells (low right), and late apoptosis and death
cells were defined as Annexin V-positive, PI-positive cells (up right)
Cancer Chemother Pharmacol (2010) 65:481–489485
123

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for 24 h, and the expressions of PARP and pro-caspase3
were observed after cells were treated for 48 h. As shown
in Fig. 5, 5-FU combined with oroxylin A significantly
decreased the protein level of Bcl-2, whereas COX-2 and
P53 protein levels were increased compared with 5-FU
single group. PARP, a 112 kDa protein, is a substrate for
caspases and cleaved during apoptosis to an 89 kDa frag-
ment. Oroxylin A or 5-FU alone activated caspase-3 and
induced PARP cleavage and the combined effect was more
significant than single treatment.
Inhibitory effect of oroxylin A or 5-FU or the
combination on the growth of H22murine solid tumor
We further examined the effects of 5-FU combined with
oroxylin A on the growth of H22murine solid tumor in
vivo. As shown in Fig. 6, combination group presented
strongest inhibitory effect on the growth of H22murine
solid tumor. As shown in the curves of tumor growth
volumes (Fig. 6a), all treated groups showed inhibitory
effects starting from the fifth day. At the seventh day, the
mean volume of the tumors in control group reached
1,588 mm3, while oroxylin A group and 5-FU group were
at 1,311 mm3and 1,096 mm3respectively, while that in
the drug-combination group was smallest with 720 mm3,
which was about one half of the data of the control group
(P\0.05 compared with control). The same trend is
shown in the tumor weight figure (Fig. 6b). In the drug-
combined group, the average tumor weight was the
smallest (0.66 g) and the inhibitory rate reached 59.67%
(P\0.01 compared with control), whereas those of mice
treated with 5-FU or oroxylin A alone were 34.71%
(P\0.05 compared with control) and 14.72% (P[0.05
compared withcontrol),respectively.
showed that the anti-tumor effect of oroxylin A combined
with 5-FU was superior to that of either single drug
treatment.
Theseresults
Discussion
At present, the most effective treatment for patients with
HCC is the surgical resection of hepatic lesions. Local
therapeutic approaches, such as transcatheter arterial
embolization [3], percutaneous transhepatic ethanol injec-
tion [7], microwave coagulation [11], and radiofrequency
ablation [25], are also effective. However, these therapies
are not sufficient for patients with advanced HCC, for
whom surgery is often not suitable and whose 5-year sur-
vival rate is extremely low [17]. For patients with advanced
HCC, the clinical responses of most anticancer drugs were
insufficient, and several combination chemotherapies have
been tried. In clinic, 5-FU combined with cisplatin, FAM
(5-FU ? adriamycin ? mitomycin) and BAF (5-FU ?
adriamycin ? bleomycetin) have been used, but the effect
is not satisfied. Therefore, to find more effective combined
therapeutic schedule becomes eager. In recent years, the
interest in exploiting traditional medicines for prevention
or treatment of tumors has increased.
In our study, the inhibitory effects of 5-FU combined
with oroxylin A on HCC cells in vitro and in vivo were
investigated. We found that oroxylin A could increase the
sensitivity of HCC cells to 5-FU. In vitro (Fig. 2), by the
method of Chou and Talalay [5], when Fa[0.075, CI\1,
oroxylin A in combination with 5-FU presented synergistic
effect on HepG2 cells when the inhibitory rate was higher
than 7.5%. The IC50 of 5-FU was reduced to about 20%
when combined with oroxylin A. The inhibitory rate on
H22murine solid tumor in vivo was 59.67% in the drug-
combination group, which was higher than either mono-
therapy (Fig. 6). While the dosing of oroxylin A in mice is
1,000 mg/kg which indicates a low bioavailability. In our
next work, we would make the drug to injectable
**
**
0
1
2
3
4
5
6
Control Oroxylin A
(50µM)
5-FU
(750µM)
Combination
TS mRN Alevel
**
**
**
0
1
2
3
4
5
6
7
8
9
10
11
Control Oroxylin A
(50µM)
5-FU
(750µM)
Combination
DPD mRNA level
##
##
Fig. 4 Changes of mRNA levels of TS and DPD in HepG2 cells. The
mRNA expression of TS and DPD was detected by real-time PCR.
HepG2 cells were treated with oroxylin A and 5-FU either in
combination or alone for 48 h. Results represent mean values of three
experiments ± SEM, *P\0.05 compared with control, **P\0.01
compared with control,##P\0.01 combination compared with 5-FU
group
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preparation, which would possess higher bioavailability in
vivo for future clinical application.
Furthermore, the apoptosis induced by oroxyloin-A
(50 lM) and 5-FU (750 lM) was examined by DAPI
staining and flow cytometry analysis. The concentrations
were selected based on determination of the IC50 for both
compounds. By DAPI staining (Fig. 3a), more apoptosis
events were observed in the combination group after HepG2
cells were incubated with drugs for 48 h. By Annexin V/PI
staining (Fig. 3b), the highest early apoptotic rate appeared
in the combination group at 26.3%, which was higher than
17.5% in oroxylin A group and 21.0% in 5-FU group.
The above results encouraged further research on the
mechanism of this synergistic effect. 5-FU is converted in
PARP
Pro-caspase3
β-actin
Cleaved PARP
P53/β-actin
Oroxylin A -- 50µM -- 50µM
5-FU -- -- 750µM 750µM
Oroxylin A -- 50µM -- 50µM
5-FU -- -- 750µM 750µM
Oroxylin A -- 50µM -- 50µM
5-FU -- -- 750µM 750µM
Oroxylin A -- 50µM -- 50µM
5-FU -- -- 750µM 750µM
Oroxylin A -- 50µM -- 50µM
5-FU -- -- 750µM 750µM
Bcl-2/β-actin
**
0
0.2
0.4
0.6
0.8
1
1.2
Pro-caspase3/β-actin
Oroxylin A -- 50µM -- 50µM
5-FU -- -- 750µM 750µM
Oroxylin A -- 50µM -- 50µM
5-FU -- -- 750µM 750µM
COX-2
P53
Bcl-2
β-actin
COX-2/β-actin
**
**
**
**
**
**
**
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
PARP/β-actin
CleavedPARP/β-actin
**
**
0
0.2
0.4
0.6
0.8
1
1.2
**
**
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
(A)(B)
(C)(F)
(G)(D)
(E)
Fig. 5 Effects of oroxylin A
and 5-FU combination on the
expression of apoptotic-related
proteins. HepG2 cells were
incubated with either oroxylin A
(50 lM) or 5-FU (750 lM)
alone or dual treatment.
Western blot assay was used to
examine COX-2, Bcl-2, and P53
expression after cells incubated
for 24 h, and PARP and pro-
caspase3 expression after cells
were treated for 48 h.
*P\0.05 compared with
control, **P\0.01 compared
with control
Cancer Chemother Pharmacol (2010) 65:481–489487
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tumor cells to FdUMP, which forms a tight covalent
complex with TS in the presence of the folate cofactor 5-
10-methylene tetrahydrofolate. And a decrease in TS levels
in tumor cells blocks DNA synthesis in dividing cells [1].
Thus, levels of TS expression in tumors have been reported
to be a critical indicator of chemosensitivity to 5-FU [15,
23]. In our study, the mRNA level of TS in the combination
group was 3.62-fold lower than that in 5-FU single group
(Fig. 4). DPD converted 5-FU accumulated into inactivated
metabolites and high DPD activity in cancer cells is an
important determinant of 5-FU response [26]. In our study,
the mRNA level of DPD in the combination group was
3.18-fold lower than that in 5-FU alone group (Fig. 4).
These results proved strongly that oroxylin A could
increase the sensitivity of HepG2 cells to 5-FU though
regulating the mRNA levels of 5-FU-related enzymes—TS
and DPD.
In addition, the changes of apoptosis-related protein
might explain the higher apoptotic rate in the drug-com-
bined group. P53 is known to induce apoptosis by tran-
scriptional repression of Bcl-2 and inhibitors of apoptosis
[32]. We detected that the expression of P53 in the
combined group was increased dramatically campared
with 5-FU group (Fig. 5). At the same time, the expres-
sion of Bcl-2 protein was down-regulated significantly
(Fig. 5). Moreover, 5-FU combined oroxylin A elevated
PARP cleavage and depressed the expression of pro-cas-
pase3. Previous reports have suggested that 5-FU could
down-regulate Bcl-2 family proteins and induces caspase
family proteins [33]. So we supposed that the combined
treatment could increase the apoptotic effects induced by
5-FU. Since P53 and Bcl-2 were the upstream events in
apoptotic pathway, we observed the changes at 24 h after
HepG2 cells being treated. The changes were not obvious
at 48 h (data not shown). Moreover, we found that at
24 h, the expression of COX-2 was decreased in the
combined group compared with oroxylin A and 5-FU
alone group. Sun et al. [24] have reported that forced
COX-2 expression significantly
induction by 5-FU through predominant inhibition of the
cytochrome c-dependent apoptotic pathway. COX-2-
mediated up-regulation of Bcl-2 suggests a potential
mechanism for reduced-apoptotic susceptibility. Based on
these data, we concluded that oroxylin A increase the
apoptosis induced by 5-FU by modulating the expressions
of apoptotic-related proteins.
Taken together, HCC in vitro and in vivo showed higher
sensitivity to 5-FU when combined with oroxylin A. The
most likely mechanism was that oroxylin A could regulate
the gene expressions of TS and DPD, the key enzymes in 5-
FU metabolic procession. Moreover, the combination of 5-
FU and oroxylin A exerted stronger apoptosis-inducing
effect than either single drug treatment, which involved the
regulation of the expression of COX-2, P53, Bcl-2, cleaved
PARP and caspase3 in HepG2 cells. All the results sug-
gested the combinational use of 5-FU and oroxylin A might
be an effective candidate in clinical treatment of HCC in
future.
attenuatesapoptosis
Acknowledgments
Technology Research and Development Program of China (863
program, No. 2003AA2Z3267), National Institutes of Health (SCORE
S06GM-008205) and Jiangsu Province Six Talent Pools in Education,
Medical care and hygiene, Electronic information, Machinery and
motor, Architecture and Agriculture Project.
This work was supported by the National High
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