中国科技论文在线 http://www.paper.edu.

cn

Available online at www.sciencedirect.com

Food and Chemical Toxicology 46 (2008) 1025–1033

www.elsevier.com/locate/foodchemtox

Cytotoxicity of fig fruit latex against human cancer cells

Jing Wang a, Xiujie Wang a,*, Shu Jiang b, Ping Lin a, Jie Zhang a, Yanrong Lu a,
Qi Wang a, Zhujuan Xiong a, Yaying Wu a, Jingjing Ren a, Hongliang Yang a
a
Division of Experimental Oncology, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University,
Chengdu 610041, Sichuan Province, PR China
b
Neourosurgery Department, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, PR China

Received 18 April 2006; accepted 26 October 2007

Abstract

Fig fruit latex (FFL) contains significant amounts of polyphenolic compounds and can serve as a source of antioxidants after human
consumption. The purpose of this study is to confirm anticancer activity of FFL against human cancer cells and to further elucidate its
mechanism of activity. Human glioblastoma, hepatocellular carcinoma, and normal liver cells were used for in vitro tests of FFL effects.
Those tests included cytotoxicity, colony formation inhibition, Brdu incorporation, acridine orange/ethidium bromide (AO/EB) staining
for apoptotic cells, cell cycle distribution through flow cytometry (FCM), and ADP-ribosyltransferase (NAD+; poly(ADP-ribose) poly-
merase)-like 1 (ADPERL1) mRNA expression through RT-PCR in response to FFL treatment. After FFL treatment, the proliferation,
colony formation, and Brdu labeling indices of cancer cells decreased (P < 0.05), while the AO/EB stained apoptotic cells increased
(P < 0.05). By FCM analysis, an increase of G0/G1 phase cell population and decrease of S and G2/M phase cells were observed
(P < 0.01), while both ADPRTL1 mRNA expression and apoptotic indices increased (P < 0.01). The findings in these studies suggested
that FFL exhibited potent cytotoxicity in some human cancer cells with little effect in normal cells at certain concentration. The mech-
anism for such effects might be associated with the inhibition of DNA synthesis, induction of apoptosis, and cell cycle arrest of cancer
cells.
Ó 2007 Elsevier Ltd. All rights reserved.

Keywords: Fig fruit latex; Human cancer cells; Growth inhibition; Apoptosis

1. Introduction considered as sources of anticancer drugs (Ferguson

Malignant cancer is the second leading cause of death
worldwide, which deteriorates the health and life of human
beings. Therefore, there is a continuing need for develop-
ment of new anticancer drugs, drug combinations, and
chemotherapy strategies by methodical and scientific explo-
ration of enormous pool of synthetic, biological, and natu-
ral products (Mukherjee et al., 2001). In light of the
continuing need for effective anticancer agents and the
association of fruit and vegetable consumption with
reduced cancer risk, edible plants are increasingly being
*
Corresponding author. Tel./fax: +86 28 85164018.
E-mail address: xiujiewang@yahoo.com (X. Wang).
et al., 2004).
In the past decades, natural products were regarded as
important sources that could produce potential chemother-
apeutic agents. Over 50% of anticancer drugs approved by
United States Food and Drug Administration since 1960
were originated from natural resources, especially from
terrestrial plants (Kim and Park, 2002; Mann, 2002).
Epidemiological studies revealed that consumption of
fruit and vegetables is associated with a decreased risk of
heart disease and cancer (Duthie et al., 2006). Experimental
investigations demonstrated that many naturally occurring
agents and plant extracts have shown antioxidant and
chemopreventive potential in a variety of bioassay systems
and animal models, having relevance to human diseases
(Aziz et al., 2003). Phytochemicals from fruits, such as

0278-6915/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2007.10.042 转载
中国科技论文在线
1026 J. Wang et al. / Food and Chemical Toxicology 46 (2008) 1025–1033
the pomegranate juice (PJ) and its ellagitannins, inhibited
proliferation and induced apoptosis in HT-29 colon cancer
and PC3 prostate cancer cells. It may have cancer-
chemopreventive as well as cancer-chemotherapeutic effects
against human cancer (Adams et al., 2006; Malik et al.,
2005). The anticancer activities of fruits might be
associated with their antioxidant properties (Son et al.,
2003).
Fig fruit (Ficus carica L.) contains high levels of poly-
phenols, flavonoids, and anthocyanins, it has been a
typical component in the health-promoting Mediterranean
diet for millennia (Solomon et al., 2006), and is cultivated
for its sweet fruits widely used both as a food and
medicine all over the world. The latex released on picking
the fruits was used to treat skin tumors and warts
(Rubnov et al., 2001). Fresh and dried fig fruits are rich
in phenolic antioxidants, and are a source of antioxidants
after human consumption (Vinson et al., 2005). Fig latex
and its component (R3) have been shown to inhibit the
growth of transplanted and spontaneous tumors in
mice (Ullman, 1952; Ullman et al., 1952), while in vitro
data showed that the extract from fig latex (resin) inhib-
ited the proliferation of some human cancer cells (Rub-
nov et al., 2001). However, the mechanism behind is
unclear.
Combinations of polyphenols naturally found in fruits
and vegetables had been suggested to be optimal for
cancer prevention (Mertens-Talcott and Percival, 2005;
Potter, 1997; Kim et al., 2006) and their anticarcinogenic
effects were well established (Mertens-Talcott and Perci-
val, 2005). Some antioxidants, such as resveratrol, are
assumed to possess cancer-preventive and cancer-thera-
peutic properties and can induce apoptosis of cancer cells
(Michels et al., 2006). Study has suggested that many che-
motherapeutic agents induce apoptosis of cancer cells
through ROS-mediated cell damage (Matthews et al.,
2006). Based on the antioxidant properties of fig latex
(Vinson et al., 2005) and its main components (R3 and
resin), which inhibited the growth of mouse tumor cells
(Ullman, 1952; Ullman et al., 1952) and some human
cancer cells in vitro (Rubnov et al., 2001), we hypothe-
sized that fig fruit latex might have anticancer activities
against some cancer cells. To confirm this hypothesis,
the inhibitory effects of fig latex on growth of human
glioma and hepatocellular carcinoma cells have been
studied and the mechanism of its activity was further
investigated.
2. Materials and methods
2.1. Preparation of fig latex
Fig fruit latex (FFL) was collected from ripe fig fruits (F. carica, in
Longquan Chengdu, China) drop-by-drop without squeezing and kept at
20 °C until processed. It was then filtered with 0.22 lm Ø microfilter,
and kept at 20 °C as stock solution (1.0 mg/ml). Solutions of final
concentrations ranging from 0.125 to 2.0 lg/ml were prepared by serial
http://www.paper.edu.cn
dilutions using RPMI-1640 (Gibco/BRL) supplemented with 10% fetal
bovine serum (Huaxi Biology Institute).
2.2. Cell line and culture
Human glioma (U251), human hepatocellular carcinoma (SMMC-
7721) and human fetal normal liver (L02) cell lines were obtained from
China Center for Type Culture Collection. Cells were maintained in
RPMI-1640 and 10% FBS with penicillin (100 U/ml) and streptomycin
(100 lg/ml; Sigma) at 37 °C, 5% CO2.
2.3. Cytotoxicity assays (MTT assay)
The cytotoxicity of FFL was determined by tetrazolium (MTT) assay
(Selvakumaran et al., 2003). Cells (2  103/well) were plated in 100 ll of
medium/well in 96-well plates. After overnight incubation, FFL was
added at various concentrations (0.125, 0.25, 0.5, 1.0, 2.0 lg/ml), 5 wells
for each concentration. After treatment with FFL for 1, 2, 3, 4, and 5 days,
20 ll of 5 mg/ml MTT (pH 4.7) was added to each well and cultivated for
another 4 h. The supernatant was removed and 100 ll DMSO was added
per well. Samples were then shaken for 15 min. The absorbance at 570 nm
was measured with microplate reader (Bio-Rad), using wells without cells
as blanks. All experiments were performed in triplicate. The effect of FFL
on the proliferation of cancer and normal cells was expressed as relatively
cell viability, using the following formula: Percent viability = OD of drug-
treated sample/OD of none treated sample)  100 (Kim et al., 2006).
2.4. Clonogenic survival determination
Cells were seeded at specified numbers (300/well for U251 cell, 400/
well for SMMC-7721 cell) in 6-well plates. Colony-forming ability was
tested following 12-day FFL treatment at 0.125, 0.25, and 0.50 lg/ml,
respectively. This was done by staining cells with 0.5% crystal violet in
absolute ethanol and counting colonies with >50 cells under dissection
microscope. As L02 cells are human fetal normal liver cells, having little
clonogenic potential compared with cancer cells, so its colony formation
assay was not performed.
2.5. BrdU incorporation in vitro
Cells (3.0  104 cells/cm2) were seeded on glass coverslips and allowed
to grow for 12 h, and then treated with 0.25 lg/ml of FFL for 48 h fol-
lowed by incubation with 20 lg/ml 5-bromo-20 -deoxyuridine in medium
for 12 h. Cells were then fixed in methanol at 20°C for 1–2 min, allowed
to air dry, then stored at 20°C until all coverslips were ready for pro-
cessing. Once ready, cells were rehydrated in PBS for 5 min followed by
immersion in 2 N HCl for 1 h at room temperature. Two times of 5 min
incubation with 0.1 M borate buffer (pH 8.5, 0.1 M boric acid, 25 mM
Na2B4O7, and 75 mM NaCl) and three times of 10 min washes in PBS
were then applied. After that, cells were incubated overnight at 4 °C with
BrdU mouse monoclonal antibody (11B5, Zymed Laboratories, USA) at a
dilution of 1:100 and then 20 min with biotylated second antibody fol-
lowed by 30 min incubation at room temperature with streptavidin/per-
oxidase. Subsequently, the sections were subjected to color reaction with
0.02% 3,3-diaminobenzidine tetrahydrochloride containing 0.005% H2O2
in PBS (pH 7.4) and were counterstained with hematoxylin lightly. The
percentage of BrdU labeled cells was determined by counting several fields
of 200 cells (in areas of the slide containing the highest BrdU labeled cells)
(Thor et al., 1999) with double blind method.
2.6. AO/EB staining for apoptotic cells
Cancer cells were seeded in 6-well plates (5  105/well) and incubated
overnight before being treated with 0.25 lg/ml FFL for 48 h. To stain
apoptotic cells, the plates were centrifuged for 5 min before l ml of AO/EB
dye mix (100 lg/ml acridine orange and 100 lg/ml ethidium bromide) was
中国科技论文在线 http://www.paper.edu.cn
J. Wang et al. / Food and Chemical Toxicology 46 (2008) 1025–1033 1027

added to each well, and cells were viewed and counted under fluorescence
microscope (Ribble et al., 2005). All experiments have been repeated three
times and minimum of 200 total cells were counted for each repeat.
2.7. Cell cycle analysis (FCM)
Cell cycle was analyzed by flow cytometry (FCM) analysis (Yuan et al.,
2004). A total of 1  107 cells were harvested from control culture and
cells treated with 0.25 lg/ml FFL for 48 h. Cells were washed twice with
PBS and fixed in 70% ice-cold ethanol for 1 h. The sample was then
concentrated by removing ethanol and treated with 1% (v/v) Triton X-100
and 0.01% RNase for 10 min at 37 °C. Cellular DNA was stained with
0.05% propidium iodide for 20 min at 4 °C in darkness. Cell cycle distri-
bution and apoptotic cells were detected with FCM (Model FACSC420,
USA). Data of more than 1  104 cells was then analyzed with the Mul-
tiCycle software package (Phoenix, USA). All data represents the results
from three independent experiments.
10 mM dNTP mix. ADPRTL1 gene (AF057160) was quantified by semi-
quantitative RT-PCR using primers: 50 -gaagaatcagtaggcagtctcg-30 and 50 -
cctggaaatagaagggtcatcc-30 with an expecting product size of 477 bp.
Primers 50 -caccacaccttctacaatgagc-30 and 50 -gtgatctccttctgcatcctgt-30 were
used to amplify b-actin, which served as an internal PCR control. PCR
products were analyzed on 1.5% agarose gel and visualized with ethidium
bromide. Image were acquired and quantified using the ChemiDocTM
XRS (Bio-Rad).
2.9. Statistical analysis
Mean data values are presented with their deviation (mean ± SEM).
All data were analyzed using analysis of variance (ANOVA), followed by
Dunnett’s test for pairwise comparison. Statistical significance was defined
as P < 0.05 for all tests.
3. Results

2.8. RT-PCR 3.1. Cell proliferation

Total RNA was isolated from the control and treated cells using the
RNeasy Minikit (Qiagen). RNA was reverse-transcribed to cDNA using The absorption values of the controls were 0.1331–
100 units of Rever Tra Ace (Toyobo, Japan), 50 lM oligo (dT) primer and 1.11020 ± 0.004–0.052 for U251 cells, 0.2347–1.2817 ±

0.125ug
A 120 0.125ug B
0.25ug * 0.25ug*
100
100 0.5ug**
Viability (% of control)

0.5ug ** 90
Viabliliy (% of control)

1.0ug ** 80 1.0ug**
80
2.0ug ** 70 2.0ug**
60
60
50

40 40
30
20 20
10
0 0
1d 2d 3d 4d 5d 1 2 3 4 5
Time (days) Time (days)

C 120 0.125ug D 120 U251

0.25ug* SMMC7721
Viability (% of control)

100
Viability (% of control)

100
0.5ug** L-02*
80 1.0ug** 80
2.0ug**
60 60

40 40

20 20

0 0
1 2 3 4 5
g

ug

ug

g
g
5u

0u
0u
25

50
12

2.
1.
0.

0.
0.

Time (days) FFL concentration

Fig. 1. Growth inhibition of FFL on human glioma, HCC and normal liver cells. Cells were seeded onto 96-well plate at 2  103/well and were treated
with FFL at different concentrations, and percentage of cell viability was determined by MTT assay after 1 d, 2, 3, 4, 5 days of treatment, respectively. A
dose- and time-dependent growth inhibition was observed at concentrations ranging from 0.125 to 2.0 lg/ml. Results are mean values ± SD of three
independent experiments performed in triplicate. (A) Growth inhibition of FFL on human glioma cells (U251); (B) growth inhibition of FFL on human
HCC cells (SMMC-7721); (C) growth inhibition of FFL on human fetal normal liver cells (L02); (D) IC50 values of cancer cells (U251, SMMC-7721) and
normal liver cell (L02) determined after the treated cells were incubated for 72 h with FFL. OD values of each treated group were compared with that of
the control at the same time point, the single asterisk (*) indicates a significant difference from the control (P < 0.05); the double asterisk (**) indicates a
very significant difference from the control (P < 0.01), one-way ANOVA, Tukey’s test. Results are mean values ± SD of independent experiments
performed in triplicate.
中国科技论文在线 http://www.paper.edu.cn
1028 J. Wang et al. / Food and Chemical Toxicology 46 (2008) 1025–1033

0.003–0.061 for SMMC-77721 cells, and 0.2477–1.2243 ±
0.009–0.061 for L02 cells on day 1, 2, 3, 4, and 5, respec-
tively. The percentages of growth inhibition of FFL at var-
ious concentrations on human cancer cells were determined
as the relatively cell viability of viable treated cells in com-
parison with viable cells of untreated controls. It showed a
dose- and time-dependent inhibitory effect on cell growth
(Fig. 1A, B and C). IC50 was determined to be 0.8 lg/ml
for U251 cells, 0.25 lg/ml and 0.8 lg/ml for SMMC-
77721 cells and L02 normal liver cells, respectively. After
FFL treatment for 72 h (Fig. 1D), the cell viability below
20% was reached at 1 lg/ml on day 4 in U251 cells, at
0.5 lg/ml on day 2 in SMMC7721 cells and at 2 lg/ml
on day 3 in L02 cells.
3.2. Colony formation
Untreated glioma cells produced 39.75 ± 2.25 colonies,
and the colony numbers were suppressed to 10.50 ± 0.65
(P < 0.05), 2.0 ± 0.71 and zero (P < 0.01), while untreated
HCC cells produced 214.50 ± 2.72 colonies, and the colony
numbers were suppressed to 77.50 ± 3.86 (P < 0.05),
1.75 ± 0.85 (P < 0.01) and zero with 0.125, 0.25, 0.50 lg/
ml FFL treatment. A dose-dependent colony-forming
inhibitory effect was observed (Fig. 2).
3.3. BrdU incorporation
The inhibitory effect of FFL on glioma, HCC and nor-
mal liver cells were further confirmed using BrdU incorpo-
ration into the untreated and treated cells in vitro (Fig. 3).
BrdU labeled cells in the untreated glioma, HCC, and
normal liver cells were 53.95 ± 1.3%, 61.12 ± 1.6% and
66.67 ± 1.1%, respectively. With 48-h treatment of
0.25 lg/ml FFL, BrdU labeled cells were 32.30 ± 1.4%,
48.74 ± 1.0% and 65.18 ± 1.3%, respectively for glioma,
HCC, and normal liver cells. Statistical significances were
found in untreated and treated cancer cells (P < 0.05),
and no significance in normal liver cells (P > 0.05).
3.4. Apoptosis
Apoptotic cells in untreated human glioma, HCC, and
normal liver cells were 2.0 ± 0.6%, 4.0 ± 0.4%, and
4.7 ± 0.3%, respectively, after treatment with 0.25 lg/ml
FFL for 48 h. The apoptotic cells in treated cancer cells
increased significantly (P < 0.01) while no effect on normal
liver cells was found (Fig. 4).
3.5. Cell cycle distribution
With 48-h treatment of 0.25 lg/ml FFL, the apoptotic
cells in treated glioma increased from 5.2 ± 1.1% to
13.0 ± 2.1% (P < 0.01), and from 5.2 ± 0.9% to
13.0 ± 1.7% (P < 0.01) in treated HCC cells. Cell number
in G0/G1 phase was increased significantly (P < 0.01) while
number of cells in S and G2/M phases decreased (P < 0.01).
Reverse effect of same treatment on normal liver cells was
found (P < 0.01, Fig. 5).
3.6. ADPRTL1 mRNA expression
ADPRTL1 mRNA expression of cancer cells treated
with 0.25 lg/ml FFL for 48 h increased more than 2-folds
(P < 0.05 for U251 cells, P < 0.01 for SMMC-77721 cells),
but no increase were found in normal liver cells (Fig. 6).
4. Discussion
Fresh and dried fig fruits contain significant amounts of
polyphenolic compounds, and are a source of in vivo anti-
oxidants after human consumption (Vinson et al., 2005).
While some plant extracts exhibited potential antioxidant
and anticancer properties, they also inhibited proliferation

250
A B
50 200
Colony number
Colony number

40
150
30
100 *
20
*
10 50
** **
** **
0 0
g
ol

ug

ol

ug

g
5u
5u

5u

5u
tr

tr

25
25
on

on

0.
12

12
0.

0.
0.
0.

0.
C

Fig. 2. Colony formation inhibition of FFL on human glioma and HCC cells. Cells were seeded onto 6-well plate (300/well for U251 cell, 400/well for
SMMC-7721 cell), and were treated with FFL at different concentrations, the colony number was counted under dissection microscope. A dose-dependent
colony formation inhibition was found. Results are mean values ± SD of three independent experiments performed in triplicate. (A) Colony formation
inhibition of FFL on human glioma cells (U251); (B) Colony formation inhibition of FFL on human HCC cells (SMMC-7721). The colony number of
each treated group were compared with that of the control, the single asterisk (*) indicates a significant difference from the control (P < 0.05); the double
asterisk (**) indicates a very significant difference from the control (P < 0.01), one-way ANOVA, Tukey’s test. Results are mean values ± SD of
independent experiments performed in triplicate.
中国科技论文在线 http://www.paper.edu.cn
J. Wang et al. / Food and Chemical Toxicology 46 (2008) 1025–1033 1029

G 80
70
Brdu labeled cells

60
50 * Control
40 * FFL0.25ug
30
20
10
0
1

21

2
25

L0
77
U

C
M
SM

Cell lines

Fig. 3. BrdU incorporation in vitro. Cells were seeded onto glass coverslips at an initial density of 3.0  104 cells/cm2 and allowed to grow for 24 h, and
then treated with 0.25 lg/ml FFL for 48 h. The cells were incubated with 5-bromo-20 -deoxyuridine in medium(20 lg/ml)for 12 h, and BrdU labeled cells
were detected with the method indicated in material and method. A, C, E, BrdU labeled cells in the untreated human glioma, HCC and normal liver cells;
B, D, F, BrdU labeled cells in 0.25 lg/ml FFL treated human glioma, HCC and normal liver cells decreased; G, the histogram shows that there was a
significant decrease of BrdU labeled cells in FFL treated cells in cancer cells and no effect on human normal liver cells. BrdU labeled cell number of treated
group were compared with that of the control, the single asterisk (*) indicates a significant difference from the control (P < 0.05), one-way ANOVA,
Tukey’s test. Results are mean values ± SD of independent experiments performed in triplicate.
中国科技论文在线 http://www.paper.edu.cn
1030 J. Wang et al. / Food and Chemical Toxicology 46 (2008) 1025–1033

A B

C 25
Control
** FFL0.25ug
20
Apoptotic cells (%)

15
**
10

0
U251 7721 L02

Fig. 4. Apoptosis induction of FFL on human glioma, HCC and normal liver cells. (A) Apoptotic cells in the untreated glioma cells (U251); (B) Apoptotic
cells in 0.25 lg/ml FFL treated glioma cells increased, blue arrows next to ‘‘L” point to live cells; yellow arrows next to ‘‘A” indicate apoptotic cells; and
red arrows next to ‘‘N” indicate necrotic cells; (C) the histogram shows that there was a significant increase of apoptotic cells in cancer cells and no effect
on human normal liver cells. The apoptotic cell number of treated group were compared with that of the control, the single asterisk (*) indicates a
significant difference from the control (P < 0.05); the double asterisk (**) indicates a very significant difference from the control (P < 0.01), one-way
ANOVA, Tukey’s test. Results are mean values ± SD of independent experiments performed in triplicate. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)

of multiple human cancer cells (Ju et al., 2004; Magiatis
et al., 2001). The anticancer activities might be associated
with their anti-oxidant properties (Son et al., 2003), and
therefore, it might be assumed that fig fruits have antican-
cer activity against some cancer cells. Previous studies
indicated that FFL and its constituent (R3) inhibited the
growth of transplanted and spontaneous tumors in mice
(Ullman, 1952; Ullman et al., 1952), the extract from
FFL (resin) inhibited the proliferation of some human can-
cer cells in vitro (Rubnov et al., 2001), and fig tree latex had
therapeutic effect on papillomatosis in cow (Hemmatzadeh
et al., 2003), and human warts (Bohlooli et al., 2007). How-
ever, the investigation of the anticancer activity of fig latex
against human cancer cells was limited, and there was no
report on anticancer activity of original fresh FFL. Our
study hereby confirms that the FFL has strong dose- and
time-dependent anticancer activity against human glioma
and HCC cells, with lower cytotoxicity in normal liver cells
(IC50 = 0.80 lg/ml). After treatment with FFL for 72 h
(Fig. 1D), the cell viability below 20% was reached at
0.5 lg/ml on day 2 in SMMC7721 cells and at 2 lg/ml
on day 3 in L02 cells, which suggested that HCC cells were
more sensitive to FFL than normal liver cells. It also inhib-
its the colony growth potential of cancer cells in a dose-
dependent manner in vitro. The study also shows that,
the anticancer activity of original fresh FFL is more potent
(IC50 = 0.25–0.8 lg/ml) than that of its extract, resin
(IC50 = 25 lg/ml) (Rubnov et al., 2001).
The anticancer activity of FFL against human glioma
and HCC cells might result, at least in part, from inhibition
of DNA synthesis and proliferation, and from induction of
apoptosis. Inhibition of DNA synthesis and proliferation
of cancer cells was verified by its ability to reduce BrdU
incorporation into cancer cells that correlates with
decreased cell proliferation (Milosevic et al., 2002). After
treatment with 0.25 lg/ml FFL for 48 h, proliferation of
BrdU labeled cancer cells was reduced significantly
(P < 0.05), while no significant effect was observed on nor-
mal liver cells (P > 0.05), indicating that FFL specifically
inhibited proliferation of cancer cells.
Apoptosis induction was additionally measured by
in situ EB/AO staining for apoptotic cells (Ribble et al.,
2005), flow cytometry (FCM) analysis (Yuan et al.,
2004), and RT-PCR semi-quantification of ADPRTL1
mRNA expression. After treatment with 0.25 lg/ml FFL
for 48 h, the apoptotic cancer cells increased significantly
(P < 0.01, Fig. 4). Specifically, the cell population in G0/
G1 phase increased (P < 0.01), those in S and G2/M phases
decreased (P < 0.01, Fig. 5), suggesting a growth arrest in
the G0/G1 phase of the cell cycle. Moreover, ADPRTL1
中国科技论文在线 http://www.paper.edu.cn
J. Wang et al. / Food and Chemical Toxicology 46 (2008) 1025–1033 1031

A 90
**
Control B 90 Control
C 80 Control
80 Treated **
80 70
Cell number (%)

Treated Treated

Cell number (%)

Cell number (%)
70
70 60
60 60 **
50
50 50
40 40
40
30 30
* 30 *
20 ** 20 ** 20
10 * 10 * 10 *
0 0 0

is

M
is

is

G
G

os
os

os

2/
2/

2/
0/

0/

0/
pt

pt

pt
G

G
G

G
po

po

o
Ap
A

A
Cell cycle distribution Cell cycle distribution Cell cycle distribution

Fig. 5. Cell cycle analysis of human human glioma, HCC and normal liver cells treated with FFL. Human glioma, HCC and normal liver cells were
treated with 0.25 lg/ml FFL for 48 h, both the control and treated cells were harvested and subjected to flow cytometric analysis. After FFL treatment, the
apoptotic cells and the cells in G0/G1 phase increased significantly, and decreased in S and G2/M phases, and no effect on human normal liver cells. (A)
apoptotic cells and cell cycle distribution of U251 cells after FFL treatment; (B) apoptotic cells and cell cycle distribution of SMMC-7721 cells after FFL
treatment; (C) apoptotic cells and cell cycle distribution of L02 cells after FFL treatment; (D) a representative picture of the flow cytometric scan of FFL
untreated (upper left) and treated (lower right) SMMC-7721 cells and FFL untreated (upper left) and treated (lower right) L02 cells. The cell numbers in
sub G1, G0/G1, S and G2/M phases of treated group were compared with those of the control, the single asterisk (*) indicates a significant difference from
the control (P < 0.05); the double asterisk (**) indicates a very significant difference from the control (P < 0.01), one-way ANOVA, Tukey’s test. Results
are mean values ± SD of independent experiments performed in triplicate.

expression increased more than 2-folds (Fig. 6) with no none IIA-induced apoptosis of promyelocytic leukemic
effect on normal liver cells was found. cells (HL60) was accompanied by the specific proteolytic
ADP-ribosyltransferase (ADPRT) gene encodes a zinc- cleavage of poly(ADP-ribose) polymerase (PARP) and
finger DNA-binding protein, poly(ADP-ribose) polymer- the activation of caspase-3, a major component in
ase-1 (PARP-1), which modifies various nuclear proteins apoptotic cell death mechanism (Yoon et al., 1999). In
by poly(ADP-ribosyl) action and functions as a key apoptosis of human breast cells induced with tanshinone
enzyme in the base excision repair pathway. Altered IIA and litchi fruit pericarp extract, ADP-ribosyltrans-
ADPRT/PARP-1 enzyme function was associated with ferase (NAD+; poly(ADP-ribose) polymerase)-like 1
response to oxidative damage, correlating with the severity (ADPRTL1) expression increased significantly ( Wang
of genotoxic stress and this determines that cellular et al., 2005, 2006).
response (Griesenbeck et al., 1997; Lockett et al., 2004; Combined with the findings in this study that showed
Koh et al., 2005). PARP plays a facilitating role in t process the apoptotic cells in FFL treated cancer cells increased sig-
of apoptosis, early PARP activation may assist the nificantly (P < 0.01, Figs. 4 and 5), and that the expression
apoptotic cascade, it is activated at an intermediate stage of ADPRTL1 increased more than 2 folds, it was suggested
of apoptosis and is then cleaved and inactivated at a late that FFL inhibited the growth of human cancer cells
stage by apoptotic proteases, namely caspase-3/CPP-32/ through apoptosis induction. Its mechanism might be
Yama/apopain and caspase-7 (Decker and Muller, 2002; involved in up-regulated expression of ADPRTL1, result-
Virag, 2005), thus inhibition of ADP-ribosyltrabsferase sig- ing in apoptotic death of cancer cells.
nificantly reduces its ability to induce apoptosis of cancer In conclusion, the potential anticancer activity of origi-
cells (Hatip-Al-Khatib et al., 2004). For example, tanshi- nal fresh FFL against human glioma and HCC cells in vitro
中国科技论文在线 http://www.paper.edu.cn
1032 J. Wang et al. / Food and Chemical Toxicology 46 (2008) 1025–1033

A M U251 SMMC-7721 L-02

C T C T C T

700bp β-actin

500bp

B 6
** Control
5
RNA level (1β/-actin)

FFL0.25 ug/ml
4

2
*
1

0
U251 7721 L-02

Fig. 6. ADPRTL1 mRNA expressions of human glioma, HCC and normal liver cells. Human glioma, HCC and normal liver cells treated with 0.25 lg/ml
FFL for 48 h. (A) a representative gel image of ADPRTL1 mRNA confirmed by RT-PCR. The amplified fragments were quantified using b-actin for the
normalization; (B) the histogram shows that there were significant increases of ADPRTL1 mRNA expression in FFL treated cancer cell, no effect on
human normal liver cells, The density of ADPRTL1 RNA/b-actin RNA of treated group were compared with that of the control, the single asterisk (*)
indicates a significant difference from the control (P < 0.05); the double asterisk (**) indicates a very significant difference from the control (P < 0.01), one-
way ANOVA, Tukey’s test. Results are mean values ± SD of independent experiments performed in triplicate.

was investigated for the first time in this study. The results
demonstrated that FFL has a strong antiproliferation effect
by inducing apoptotic cell death, inhibiting DNA synthesis
of cancer cells, and causing G0/G1 phase arrest of cancer
cells. Some constituents from FFL might serve as a novel
and potent anticancer agents, and need to be investigated
further, especially with animal tumor models to confirm
its anticancer activityin vivo. In addition, as there are
2.5% of atopic individuals sensitive to Ficus benjamina latex
(Hemmer et al., 2004), allergic reactions to FFL should be
considered in animal and/or clinical studies.
References
Adams, L.S., Seeram, N.P., Aggarwal, B.B., Takada, Y., Sand, D., Heber,
D., 2006. Pomegranate juice, total pomegranate ellagitannins, and
punicalagin suppress inflammatory cell signaling in colon cancer cells.
Journal of Agricultural and Food Chemistry 8, 980–985.
Aziz, M.H., Kumae, R., Ahmad, N., 2003. Cancer chemoprevention by
resveratrol: in vitro and in vivo studies and the underlying mechanisms
(review). International Journal of Oncology 23, 17–28.
Bohlooli, S., Mohebipoor, A., Mohammadi, S., Kouhnavard, M.,
Pashapoor, S., 2007. Comparative study of fig tree efficacy in the
treatment of common warts (Verruca vulgaris) vs. cryotherapy.
International Journal of Dermatology 46 (5), 524–526.
Decker, P., Muller, S., 2002. Modulating poly(ADP-ribose) polymerase
activity: potential for the prevention and therapy of pathogenic
situations involving DNA damage and oxidative stress. Current
Pharmaceutical Biotechnology 3, 275–283.
Duthie, S.J., Jenkinson, A.M., Crozier, A., Mullen, W., Pirie, L., Kyle, J.,
Yap, L.S., Christen, P., Duthie, G.G., 2006. The effects of cranberry
juice consumption on antioxidant status and biomarkers relating to
heart disease and cancer in healthy human volunteers. European
Journal of Nutrition 45, 113–122.
Ferguson, P.J., Kurowska, E., Freeman, D.J., Chambers, A.F., Koropat-
nick, D.J., 2004. A flavonoid fraction from cranberry extract inhibits
proliferation of human tumor cell lines. The Journal of Nutrition 134,
1529–1535.
Griesenbeck, J., Oei, S.L., Mayer-Kuckuk, P., Ziegler, M., Buchlow, G.,
Schweiger, M., 1997. Protein–protein interaction of the human
poly(ADP-ribosyl)transferase depends on the functional state of the
enzyme. Biochemistry 36, 7297–7304.
Hatip-Al-Khatib, I., Iwasaki, K., Chung, E.H., Egashiea, N., Mishima,
K., Fujiwara, M., 2004. Inhibition of poly(ADP-ribose) polymerase
and caspase-3, but not caspase-1, prevents apoptosis and improves
spatial memory of rats with twice-repeated cerebral ischemia. Life
Sciences 75, 1967–1978.
Hemmatzadeh, F., Fatemi, A., Amini, F., 2003. Therapeutic effects of
fig tree latex on bovine papillomatosis. Journal of Veterinary
Medicine Series B Infectious Diseases and Veterinary Public Health
50, 473–476.
Hemmer, W., Focke, M.GötzM., Jarisch, R., 2004. Sensitization to Ficus
benjamina: relationship to natural rubber latex allergy and identifica-
tion of foods implicated in the Ficus-fruit syndrome. Clinical and
Experimental Allergy 34, 1251–1258.
Ju, E.M., Lee, S.E., Hwang, H.J., Kim, J.H., 2004. Antioxidant and
anticancer activity of extract from Betula platyphylla var. japonica.
Life Sciences 74, 1013–1026.
Kim, J., Park, E.J., 2002. Cytotoxic anticancer candidates from natural
resources. Current medicinal chemistry. Anti-cancer Agents 2, 485–
537.
中国科技论文在线
J. Wang et al. / Food and Chemical Toxicology 46 (2008) 1025–1033
Kim, M.J., Kim, Y.J., Park, H.J., Chung, J.H., Leem, K.H., Kim, H.K.,
2006. Apoptotic effect of red wine polyphenols on human colon cancer
SNU-C4 cells. Food and Chemical Toxicology 44, 898–902.
Koh, D.W., Dawson, T.M., Dawson, V.L., 2005. Mediation of cell death by
poly(ADP-ribose) polymerase-1. Pharmacological Research 52, 5–14.
Lockett, K.L., Hall, M.C., Xu, J., Zheng, S.L., Berwick, M., Chuang,
S.C., Clark, P.E., Cramer, S.D., Lohman, K., Hu, J.J., 2004. The
ADPRT V762A genetic variant contributes to prostate cancer suscep-
tibility and deficient enzyme function. Cancer Research 64, 6344–6348.
Magiatis, P., Pratsinis, H., Kalpoutzakis, E., Konstantinidou, A., Davaris,
P., Skaltsounis, A.L., 2001. Hydrolyzable tannins, the active constit-
uents of three Greek Cytinus taxa against several tumor cell lines.
Biological & Pharmaceutical Bulletin 24, 707–709.
Malik, A., Afaq, F., Sarfaraz, S., Adhami, V.M., Syed, D.N., Mukhtar,
H., 2005. Pomegranate fruit juice for chemoprevention and chemo-
therapy of prostate cancer. Proceedings of the National Academy of
Sciences of the United States of America 102, 14813–14818.
Mann, J., 2002. Natural products in cancer chemotherapy: past, present
and future. Nature Reviews. Cancer 2, 143–148.
Matthews, G.M., Howarth, G.S., Butlter, R.N., 2006. Nutrient and
antioxidant modulation of apoptosis in gastric and colon cancer cells.
Cancer Biology and Therapy 5, 569–572.
Mertens-Talcott, S.U., Percival, S.S., 2005. Ellagic acid and quercetin
interact synergistically with resveratrol in the induction of apoptosis
and cause transient cell cycle arrest in human leukemia cells. Cancer
Letters 218, 141–151.
Michels, G., Watjen, W., Weber, N., Niering, P., Chovolou, Y.,
Kampkotter, A., Proksch, P., Kahl, R., 2006. Resveratrol induces
apoptotic cell death in rat H4IIE hepatoma cells but necrosis in C6
glioma cells. Toxicology 225, 173–182.
Milosevic, J., Kanazir, S., Medic-Mijacevic, L., Pejanovic, V., Stokic, Z.,
Konjevic, G., Rakic, L., Ruzdijic, S., 2002. Sulfinosine-induced cell
growth inhibition and apoptosis in human lung carcinomas in vitro.
Investigational New Drugs 20, 229–240.
Mukherjee, A.K., Basu, S., Sarkar, N., Ghosh, A.C., 2001. Advances in
cancer therapy with plant based natural products. Current medicinal
Chemistry 8, 1467–1486.
Potter, J.D., 1997. Cancer prevention: epidemiology and experiment.
Cancer Letters 114, 7–9.
Ribble, D., Goldstein, N.B., David, A., Norris, D.A., Shellman, Y.G.,
2005. A simple technique for quantifying apoptosis in 96-well plates.
BMC Biotechnology 5, 1–7.
Rubnov, S., Kashman, Y., Rabinowitz, R., Schlesinger, M., Mechoulam,
R., 2001. Suppressors of cancer cell proliferation from fig (Ficus carica)
resin: isolation and structure elucidation. Journal of Natural Products
64, 993–996.
http://www.paper.edu.cn
1033
Selvakumaran, M., Pisarcik, D.A., Bao, R., Yeung, A.T., Hamilton, T.C.,
2003. Enhanced cisplatin cytotoxicity by disturbing the nucleotide
excision repair pathway in ovarian cancer cell lines. Cancer Research
63, 1311–1316.
Solomon, A., Golubowicz, S., Yablowicz, Z., Grossman, S., Bergman, M.,
Gottlieb, H.E., Altman, A., Kerem, Z., Flaishman, M.A., 2006.
Antioxidant activities and anthocyanin content of fresh fruits of
common fig (Ficus carica L.). Journal of Agricultural and Food
Chemistry 54, 7717–7723.
Son, Y.O., Kim, J., Lim, J.C., Chung, Y., Chung, G.H., Lee, J.C., 2003.
Ripe fruit of Solanum nigrum L. inhibits cell growth and induces
apoptosis in MCF-7 cells. Food and Chemical Toxicology 41, 1421–
1428.
Thor, A.D., Liu, S., Moore 2nd, D.H., Edgerton, S.M., 1999. Comparison
of mitotic index, in vitro bromodeoxyuridine labeling, and MIB-1
assays to quantitate proliferation in breast cancer. Journal of Clinical
Oncology 17, 470–477.
Ullman, S.B., 1952. The inhibitory and necrosis-inducing effects of the
latex of Ficus carica L. on transplanted and spontaneous tumors.
Experimental Medicine and Surgery 10, 26–49.
Ullman, S.B., Clark, G.M., Roan, K.M., 1952. The effects of the fraction
R3 of the latex of ficus carica L. on the tissues of mice bearing
spontaneous mammary tumors. Experimental Medicine and Surgery
10, 287–305.
Vinson, J.A., Zubik, L., Bose, P., Samman, N., Proch, J., 2005. Dried
fruits: excellent in vitro and in vivo antioxidants. Journal of the
American College Nutrition 24, 44–50.
Virag, L., 2005. Structure and function of poly(ADP-ribose) polymerase-
1: role in oxidative stress-related pathologies. Current Vascular
Pharmacology 3, 209–214.
Yoon, Y., Kim, Y.O., Jeon, W.K., Park, H.J., Sung, H.J., 1999.
Tanshinone IIA isolated from Salvia miltiorrhiza BUNGE induced
apoptosis in HL60 human premyelocytic leukemia cell line. Journal of
Ethnopharmacology 68, 121–127.
Yuan, S.L., Wei, Y.Q., Wang, X.J., Xiao, F., Li, S.F., Zhang, J., 2004.
Growth inhibition and apoptosis induction of tanshinone II-A on
human hepatocellular carcinoma cells. World Journal of Gastroenter-
ology 10, 2024–2028.
Wang, X., Wei, Yu., Yuan, S., Liu, G., Lu, Y., Zhang, J., Wang, W., 2005.
Potential anticancer activity of tanshinone IIA against human breast
cancer. International Journal of Cancer 116, 799–807.
Wang, X., Yuan, S., Wang, J., Lin, P., Liu, G., Lu, Y., Zhang, J., Wang,
W., Wei, Y., 2006. Anticancer activity of litchi fruit pericarp extract
against human breast cancer in vitro and in vivo. Toxicology and
Applied Pharmacology 215, 168–178.