Toxicology and Applied Pharmacology 215 (2006) 168 – 178

www.elsevier.com/locate/ytaap

Anticancer activity of litchi fruit pericarp extract against human

breast cancer in vitro and in vivo
Xiujie Wang a,⁎, Shulan Yuan a , Jing Wang a , Ping Lin a , Guanjian Liu b , Yanrong Lu a ,
Jie Zhang a , Wendong Wang c , Yuquan Wei d,⁎
a
Division of Experimental Oncology, National Key Laboratory of Biotherapy, West China Hospital, Sichuan University,
Chengdu 610041, Sichuan Province, P.R. China
b
Department of Clinical Epidemiology, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, P.R. China
c
Department of Pathology of Public Health School, Sichuan University, Chengdu 610041, Sichuan Province, P.R. China
d
Division of Biotherapy, National Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, P.R. China
Received 8 October 2005; revised 23 January 2006; accepted 8 February 2006
Available online 23 March 2006

Abstract

Litchi fruit pericarp (LFP) extract contains significant amounts of polyphenolic compounds and exhibits powerful antioxidative activity against
fat oxidation in vitro. The purpose of this study is to confirm the anticancer activity of LFP extract on human breast cancer in vitro and in vivo, and
to elucidate the mechanism of its activity. Human breast cancer cells were tested in vitro for cytotoxicity, colony formation inhibition, BrdU
incorporation, and gene expression profiling after treatment with LFP extract. Seven nude mice bearing human breast infiltrating duct carcinoma
orthotopically were tested for its anticancer activity and expression of caspase-3 in vivo by oral administration of 0.3% (0.3 mg/ml) of LFP water-
soluble crude ethanolic extract (CEE) for 10 weeks. LFP extract demonstrated a dose- and time-dependent inhibitory effect on cell growth
(IC50 = 80 μg/ml), and it significantly inhibited colony formation and BrdU incorporation of human breast cancer cells. Oligonucleotide
microarray analysis identified 41(1.22%) up-regulated and 129 (3.84%) down-regulated genes after LFP water-soluble CEE treatment; the
predominantly up-regulated genes were involved in various biological functions including cell cycle regulation and cell proliferation, apoptosis,
signal transduction and transcriptional regulation, and extracellular matrix/adhesion molecules; and down-regulated genes were mainly associated
with adhesion, invasion, and malignancy of cancer cells. A 40.70% tumor mass volume reduction and significant increase of casepase-3 protein
expression were observed in vivo experiment. The findings in this study suggested that LFP extract might have potential anticancer activity on
both ER positive and negative breast cancers, which could be attributed, in part, to its DNA damage effect, proliferating inhibition and apoptosis
induction of cancer cells through up-regulation and down-regulation of multiple genes involved in cell cycle regulation and cell proliferation,
apoptosis, signal transduction and transcriptional regulation, motility and invasiveness of cancer cells; ADP-ribosyltransferase (NAD+; poly
(ADP-ribose) polymerase)-like 1 (ADPRTL1), Cytochrome P450, subfamily I (CYP1A1) and Hyaluronan-mediated motility receptor (HMMR)
might be the main molecular targets at which LFP water-soluble CEE acted.
© 2006 Elsevier Inc. All rights reserved.

Keywords: Litchi fruit pericarp extract; Anticancer activity; Breast cancer; Growth inhibition; Apoptosis

Introduction

⁎ Corresponding authors. X. Wang is to be contacted at Division of
Experimental Oncology, National Key Laboratory of Biotherapy, West China
Hospital, Sichuan University, Chengdu 610041, Sichuan Province, P.R. China.
Fax: +86 28 85164029. Y. Wei, Division of Biotherapy, National Key
Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu
610041, Sichuan Province, P.R. China. Fax: +86 28 85164059.
E-mail addresses: xiujiewang@yahoo.com (X. Wang),
yuquanwei@mail.sc.cninfo.net (Y. Wei).
0041-008X/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.taap.2006.02.004
Breast cancer is one of the main life-threatening diseases
that a woman may have to face during her lifetime
(Angelopoulos et al., 2004). The increasing incidence of
breast neoplasia reported over the last a few decades has led
to development of new anticancer drugs, drug combinations,
and chemotherapy strategies by methodical and scientific
exploration of enormous pool of synthetic, biological, and
 X. Wang et al. / Toxicology and Applied Pharmacology 215 (2006) 168–178
natural 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 considered as
sources of anticancer drugs (Ferguson et al., 2004); there is a
large amount of scientific evidence showing that fruits and
vegetables lower the risk of cancer (Chen et al., 2004), and
medicinal plants constitute the main source of new pharma-
ceuticals and healthcare products, including medications for
ethnoveterinary medicine (Ivanova et al., 2005). Recently,
cancer chemoprevention with strategies using foods and
medicinal herbs has been regarded as one of the most visible
fields for cancer control (Jo et al., 2004); however, whether
fruit, vegetable, and antioxidant micronutrient consumption is
associated with a reduction in breast cancer incidence remains
unresolved (Gaudet et al., 2004).
Epidemiological studies suggested that antioxidant supple-
ments might reduce the risk of breast cancer recurrence or breast
cancer-related mortality (Fleischauer et al., 2003), and consum-
ing food and beverages rich in polyphenols (e.g., catechins,
flavones, and antocyanines) is associated with a lower incidence
of cancers (Naasani et al., 2003). Experimental investigations
demonstrated that many naturally occurring agents and plant
extracts have shown antioxidant and anticancer potential in a
variety of bioassay systems and animal models, having
relevance to human disease (Aziz et al., 2003), e.g., Crude
methanolic extract (CME) from the pericarp of Garcinia
mangostana (family Guttiferae) has antiproliferative, apoptotic,
and antioxidative activities against human breast cancer cell line
in vitro (Primchanien et al., 2004). The antioxidant and
anticancer activity of the extracts from medical plants and
herbs was associated with their components of phenolic
compounds; the major types of phenolic compounds included
phenolic acids, flavonoids, tannins, coumarins, lignans, qui-
nones, stilbenes, and curcuminoids (Cai et al., 2004).
Litchi (Litchi chinensis, Sapindaceae) is a tree that originates
from China and is cultivated for its sweet fruits all over the
world in warm climates (Gontier et al., 2000). Pharmacological
studies showed the petroleum ether extract of leaves of the plant
Litchi chinensis Gaertn possess anti-inflammatory, analgesic,
and antipyretic activity without toxicity (Besra et al., 1996).
Litchi fruit pericarp (LFP) contains significant amounts of
polyphenolic compounds, the principal characteristic of the
polyphenolic compounds is their ortho-diphenolic structure,
which gives them high oxidability; the major components of
fresh LFP extract were condensed tannins (polymeric proantho-
cyanidins), epicatechin, and procyanidin A2 (Sarni-Manchado
et al., 2000). The main components of mature and premature
LFP extract were phenolic compound and flavonoids, which
exhibited powerful antioxidative activity against fat oxidation in
vitro (Zheng et al., 2003).
Based on the main components of LFP extract and its
antioxidant properties (Sarni-Manchado et al., 2000; Zheng et
al., 2003), we hypothesized that LFP extract may have
anticancer activities against some cancer cell lines in vitro and
animal models in vivo. However, there are no such reports. To
confirm this hypothesis, the inhibitory effect of LFP extract on
169
the growth of human breast cancer cells in vitro and the growth
of human breast infiltrating ductal carcinoma (IDC) in vivo, and
the mechanism of its activity were investigated in this
experimental study.
Materials and methods
Reagents. RPMI-1640 was purchased from GIBCO/BRL Invitrogen
(Caithershurg, MD). Fetal bovine serum (FBS) was purchased from Huaxi
Biology Institute (Chengdu, People's Republic of China). Trypsin, methylthia-
zolyldiphenyl-tetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO)
were purchased from the Sino-American Biotechnology Company of Beijing
(Beijing, People's Republic of China). 5-Bromo-2-deoxy-uridine (BrdU) was
purchased from Roche (Nutley, NJ), and S-P immunohistochemical staining kit
(SP9001) was purchased from Beijing Zhongshan Biological Technology Ltd.
(Beijing, People's Republic of China). All of other chemicals and reagents were
obtained from Sigma (St. Louis, MO).
Preparation of extracts. Mature Baila litchi (L. chinensis, Sapindaceae in
Guangdong, China) fruit pericarp was collected, dried at room temperature
naturally, and powdered. The powdered material (100 g) was extracted with 95%
ethanol (800 ml, two times) for 48 h at room temperature, blended with magnetic
force stirrer continuously. The extracts were filtered and concentrated to remove
the solvent at 75 °C for 4 h and freeze dried, and more than 22.0 g of crude
ethanolic extract (CEE) was yielded eventually, the percentage yield was higher
than 22.0%. Of the extract, 60% was water-soluble, 40% was soluble in ethanol
and acetone.
Cell line and culture. Human breast cancer MCF-7 (GDC055) and MDA-
MB-231(HTB-26) cell lines were obtained from China Center for Type Culture
Collection (Nanjing, People's Republic of China). The cells were maintained in
RPMI-1640 supplemented with 10% FBS, penicillin (100 U/ml), and
streptomycin (100 μg/ml) in a humidified atmosphere of 50 μg/ml CO2 at 37 °C.
In vitro assay for cytotoxic activity (MTT assay). The cytotoxicity of LFP
water-soluble CEE on both MCF-7 and MDA-MB-231 cells was determined
by the MTT assay (Selvakumaran et al., 2003). Cells (3 × 103/well) were
plated in 100 μl of medium/well in 96-well plates (Costar Corning, Rochester,
NY). After incubation overnight, LFP water-soluble CEE was added in
various concentrations (20, 40, 80, 160, 320 μg/ml) without cytotoxicity to
human normal liver cell line L-02 (reported in elsewhere); 5 wells were
included in each concentration. After treatment with LFP water-soluble CEE
for 1, 2, 3, 4, and 5 days, 20 μl of 5 mg/ml MTT (pH 4.7) was added per
well and cultivated for another 4 h, the supernatant fluid was removed, 100 μl
DMSO was added per well and shaken for 15 min. The absorbance at 570 nm
was measured with a microplate reader (Bio-Rad, Richmond, CA), using
wells without cells as blanks. All experiments were performed in triplicate.
The effect the water-soluble CEE from LFP on the proliferation of human
breast cancer cells was expressed as the % cytoviability, using the following
formula: %cytoviability = A570 of treated cells / A570 of control
cells × 100%.
Clonogenic survival determination. MCF-7 and MDA-MB-231 cells were
assayed for colony-forming ability by replating them in specified numbers
(300–400/well) in 6-well plates and treated with 40, 80, and 160 μg/ml of LFP
water-soluble CEE, respectively. After 12 days of incubation, the cells were
stained with 0.5% crystal violet in absolute ethanol and colonies with > 50 cells
were counted under dissection microscope.
BrdU incorporation in vitro. Cells were seeded onto glass coverslips at an
initial density of 4.0 × 104 cells/cm2 and allowed to grow for 12 h, and then
treated with 100 μg/ml of LFP water-soluble CEE for 48 h. The cells were
incubated with BrdU (20 μg/ml)for 12 h. At the appropriate time, the cells were
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 processing. The cells were rehydrated
in PBS for 5 min followed by immersion in 2 N HCl for 1 h at room temperature.
The cells were incubated in 0.1 M borate buffer (pH 8.5, 0.1 M boric acid,
170 X. Wang et al. / Toxicology and Applied Pharmacology 215 (2006) 168–178
25 mM Na2B4O7, and 75 mM NaCl) twice for 5 min each, followed by three
times of 10 min washes in PBS. Next, cells were incubated with BrdU mouse
monoclonal antibody (11B5, Zymed Laboratories, USA) at a dilution of 1:100,
overnight at 4 °C, with biotylated second antibody for 20 min, and with
streptavidin/peroxidase for 30 min at room temperature. 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. A percentage of BrdU-labeled cells
were determined by counting several fields of 200 cells (in areas of the slide
containing the highest labeling cells) (Thor et al., 1999).
Oligonucleotide microarray analysis. Human breast cancer MCF-7 cells
were treated with 100 μg/ml of LFP water-soluble CEE for a required time
(48 h). Total RNA was extracted from the treated and untreated cells using the
Qiagen (Valencia City, CA) RNeasy Minikit. cDNA was synthesized from total
RNA using cDNA Synthesis System (Roche), cRNA probes were created from
cDNA with cy3-dUTP using MEGAscript T7 Kit (Ambion, Austin, TX) and
hybridized to HO4 ExpressChip (Mergen, San Francisco, CA) containing 3360
genes (www.mergen.ltd.com), according to the manufacturer's instructions.
After a series of washes, the hybridized slides were scanned with GMS418
Array Scanner (Affymatrix, Santa Clara, CA). The signal intensity of each pair
of spots was quantified using ImaGene4.0 software (BioDiscovery, El Segundo,
CA) by subtracting the local regional background intensity from each spot; the
two slides were normalized to each other by using the signal of global
normalization gene (Glyceraldehyde-3-phosphate dehydrogenase, GAPD)
provided by the manufacturer. Fold changes were calculated and expressed
relative to untreated control sample for each pair of spots; the results were
confirmed by duplicate spots of cRNA.
RT-PCR. To verify the microarray results, the representative up-regulated
(ADPRTL1, CYP1A1) and down-regulated (HMMR) genes in response to LFP
water-soluble CEE treatment were confirmed experimentally by semi-
quantitative RT-PCR (Morandi et al., 2006).
The primers for each gene were designed with Primers Express software
(Applied Biosystems) according to ExpressChip Gene Database of the
manufacturer (Mergen, San Francisco, CA). Primer sequences and the length
of amplified products were as follows:
ADPRTL (accession number: AF057160, product size: 477 bp)
Fw 5′-gaagaatcagtaggcagtctcg-3′ Rew 5′-cctggaaatagaagggtcatcc-3′
CYP1A1 (accession number: K03191, product size: 486 bp)
Fw 5′-acctctacaccttcaccctcatc-3′ Rew 5′- agtgctccttgaccatcttctg-3′
HMMR (accession number: U29343, product size: 390 bp)
Fw 5′- gatactaccttgcctgcttcag-3′ Rew 5′- cttagccatcatacccctcatc-3′
β-actin (accession number: BC013380, product size: 695 bp)
Fw 5′-caccacaccttctacaatgagc-3′ Rew 5′-gtgatctccttctgcatcctgt-3′
Total RNA was isolated from the control and treated cells as described
above. One microgram of total RNA was retro-transcribed to cDNA using 100
Units of Rever Tra Ace (Toyobo, Japan), 50 μM Oligo(dT) primer, and 10 mÌ
dNTP mix.
PCR amplification was performed with Taq PCR MasterMix (Tiangen
Biotech, Beijing, People's Republic of China) using the following conditions:
denaturation for 30 s at 94 °C, annealing for 30 s at 56 °C, and elongation for
45 s at 72 °C for 30 cycles.
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).
In vivo tumor growth inhibition study. To confirm the anticancer activity of
LFP water-soluble CEE on human breast IDC, an in vivo experiment was carried
out using an ER negative human breast IDC-xenografted animal model (Chen et
al., 2003). Thirteen 6-week-old female nude mice (Experimental Animal Center,
Sichuan University) were xenotransplanted with human breast infiltrating duct
carcinoma F35 orthotopically (Chen et al., 2003). The procedures involving
animals and their care were conducted in accordance with institutional guidelines
for Laboratory Animal Care of Experimental Animal Center, Sichuan University.
At 2 weeks, tumors reached 50 mm3 in volume, the tumor bearing mice were
randomized into two experimental groups. The treatment group animals (n = 7)
were fed with 0.3% (0.3 mg/ml) of LFP water-soluble CEE through drink water
ad libitum for 10 weeks, the control animals (n = 6) were without treatment. Mice
were weighted, and tumor volume was assessed by measuring two perpendicular
dimensions (long and short) using a caliper and calculated using the formula
(a × b2) / 2, where a is the larger and b is the smaller dimension of the tumor
(Zhang et al., 2003).
Immunohistochemical analysis of tumors for caspase-3 protein
expression. Tumor samples from mice were fixed in 4% paraformaldehyde for
24 h and processed conventionally. Caspase-3 protein expressions were detected
immunohitochemically with SP kit (Zymed); briefly, tissue sections were de-
paraffinized and rehydrated through graded alcohols. Antigen retrieval was
performed by microwave oven heating in 0.1 mM citrate buffer (pH 6). Then,
endogenous peroxidase activity was blocked with 0.3% H2O2, and after
treatment with normal goat serum, the tissue sections were incubated with
caspase-3 rabbit polyclonal antibody (Borster Biotechnology, Wuhan, People's
Republic of China) at a dilution of 1:100, overnight at 4 °C, with biotylated
second antibody 20 min, and with streptavidin/peroxidase 30 min at room
temperature. 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. Caspase-3
positive cells were determined by counting several fields of 200 cells, Caspase-3
labeling index (LI) was calculated using the formula Caspase-3 labeling index
(LI) = Number of activated caspase-3-positive cells / Total number of
nuclei × 100% (Duan et al., 2003).
Statistical analysis. The statistical significance of difference between control
and LFP-extract-treated groups was determined by one-way ANOVA followed
by Tukey test for multiple comparisons. Dunnett's t tests (2-sided) were
employed, as needed, and result was considered significant at P < 0.05.
Results
Cytotoxic activity of LFP water-soluble CEE against human
breast cancer cells
LFP water-soluble CEE showed a dose- and time-dependent
inhibitory effect on the growth of MCF-7 and MDA-MB-231
breast cancer cells (P < 0.05). IC50 was 80 μg/ml, and the
maximal inhibition of cell growth (>80%) was obtained at
320 μg/ml. The result of cytotoxic activity of LFP water-soluble
CEE against human breast cancer cells is shown in Fig. 1.
Inhibition of colony formation
Untreated MCF-7 and MDA-MB-231cells produced
235 ± 16 and 205 ± 12 colonies, respectively; the colony
numbers of MCF-7 cells were suppressed to 145 ± 20
(P < 0.05), 70 ± 13 (P < 0.01), and 28 ± 9 (P < 0.01) with
40, 80, and 160 μg/ml of LFP water-soluble CEE treatment,
respectively; the colony numbers of MDA-MB-231 cells were
suppressed to 118 ± 16 (P < 0.05), 62 ± 11 (P < 0.01), and 21 ± 7
(P < 0.01) after the treatment of 40, 80, and 160 μg/ml of LFP
water-soluble CEE, respectively; and a dose-dependent colony-
forming inhibition effect on both cell lines were observed
(Fig. 2).
Inhibition of cell proliferation
The inhibitory effect of LFP water-soluble CEE on MCF-7
cells was further confirmed using BrdU incorporation into the
 X. Wang et al. / Toxicology and Applied Pharmacology 215 (2006) 168–178 171

(HPRP3P), General transcription factor IIA, 2 (GTF2A2),

Splicing factor (CC1.3), HMMR, A disintegrin and metallo-
proteinase domain 9 (ADAM9), Cytochrome P450, subfamily
IIIA polypeptide 3 (CYP3A3), were down-regulated for more
than 5-fold (Table 2, Fig. 4). The range of fold regulation varied
widely with ADP-ribosyltransferase (ADPRTL1) exhibiting the
maximum up-regulation (29.25-fold) and Hyaluronan-mediated
motility receptor (RHAMM) exhibiting the maximum down-
regulation (−15.48-fold).

Confirmation of the representative genes by RT-PCR

To assess the reliability of the microarray results, 3

representative genes with increased or decreased expression
were analyzed by semi-quantitative RT-PCR using β-actin as
the reference gene to normalize expression data. A good
qualitative correlation was observed in the results obtained
by the two techniques, with all 3 genes tested showing
similar trends; however, quantitative differences in regulation
are present (Fig. 5).
Fig. 1. Inhibition of human breast cancer cell growth by LFP water-soluble CEE.
Cells were seeded onto 96-well plate at 3 × 103/well and were treated with LFP
water-soluble CEE at different concentrations, and percentage of cell viability
was determined by MTT assay after 1–5 days of treatment, respectively. (A) A
dose- and time-dependent growth inhibition of MCF-7 breast cancer cells (ER
positive) was observed at concentrations ranging from 20 to 320 μg/ml
(P < 0.05); (B) A dose- and time-dependent growth inhibition of MDA-MB-231
breast cancer cells (ER negative) was observed at concentrations ranging from
20 to 320 μg/ml (P < 0.05). Results are mean values ± SD of independent
experiments performed in triplicate.

untreated and treated (100 μg/ml) breast cancer cells in vitro

(Fig. 3). BrdU-labeled cells in LFP water-soluble CEE-treated
cells were 15.20 ± 1.30%, and lower than that in untreated
controls (33.50 ± 2.18%), a statistical significance was found
(P < 0.05).

Change of gene expression profile

Out of 3360 genes studied, 170 differential genes were

identified from oligonucleotide microarray in which gene
expression was increased or decreased more than 2-fold in
cells treated with 100 μg/ml of LFP water-soluble CEE
compared with control cells cultured under identical condition
without treatment; 41(1.22%) were up-regulated (Table 1) and
129 (3.84%) were down-regulated (Table 2). Eight genes
showed more than 3-fold up-regulation, and 38 showed more
than 3-fold down-regulation, two genes were up-regulated for
Fig. 2. Inhibition of human breast cancer cell colony formation by LFP water-
more than 5-fold, i.e., Cytochrome P450, subfamily I (aromatic
soluble CEE. Cells were seeded onto 6-well plate at 300/well and were
compound-inducible), polypeptide 1 (CYP1A1), and ADP- treated with LFP water-soluble CEE at different concentration; the colony
ribosyltransferase (NAD+; poly (ADP-ribose) polymerase)- number was counted under dissection microscope. (A) A dose-dependent
like1 (ADPRTL1); their expression level increased by 7.04-fold colony formation inhibition on MCF-7 breast cancer cells (ER positive); (B) a
and 29.25-fold, respectively (Table 1, Fig. 4), and 10 genes dose-dependent colony formation inhibition on MDA-MB-231 (ER negative)
breast cancer cells. The single asterisk (*) indicates a significant difference
including Gardner–Rasheed feline sarcoma viral (v-fgr)
from the control (P < 0.05); the double asterisk (**) indicates a very
oncogene homolog (FGR), Glucagon-like peptide 1 receptor significant difference from the control (P < 0.01), one-way ANOVA, Tukey's
(GLP1R), Baculoviral IAP repeat-containing 3 (BIRC3), test. Results are mean values ± SD of independent experiments performed in
Interleukin 3 (IL3), U4/U6-associated RNA splicing factor triplicate.
172 X. Wang et al. / Toxicology and Applied Pharmacology 215 (2006) 168–178

Fig. 3. BrdU incorporation in vitro. MCF-7 breast cancer cells were seeded onto glass coverslips at an initial density of 4.0 × 104 cells/cm2 and allowed to grow for
12 h, and then treated with 100 μg/ml of LFP water-soluble CEE for 48 h. The cells were incubated with 5-bromo-2′-deoxyuridine in medium (20 μg/ml) for 12 h, and
BrdU-labeled cells were detected with the method indicated in Materials and methods. (A) Representative photographs of BrdU-labeled cells in the untreated cells; (B)
representative photographs of BrdU-labeled cells in the treated cells decreased; (C) the histogram shows a significant decrease of BrdU-labeled cells in LFP water-
soluble CEE-treated cells, the single asterisk (*) indicates a significant difference from the control, one-way ANOVA, Tukey's test (P < 0.05).

Inhibition of tumor growth in human breast IDC-xenografted there is a significant inhibition of tumor growth in the treated
nude mice mice as compared with the control mice (Fig. 6). No evidence
of drug-related toxicity was identified in the treated animals by
The average tumor volume at the end of experiment was comparing the body weight increase, histopathological
738.8 ± 328.11 mm3 in the treated mice and 1232.8 ± changes of major organs, and blood biochemistry analysis of
424.65 mm3 in the control mice; tumor growth inhibitory rate both group animals (data not shown).
was 40.70% (P < 0.05). The tumor volume in the treated mice
increased 8.80 mm3 everyday, 1.67 times slower than that in Increase of caspase-3 protein expression in tumors
controls (14.68 mm3). The experimental finding showed that
The antibody specific for activated caspase-3 selectively
labeled the cytoplasm of cells that had morphology consistent
Table 1
List of genes up-regulated more than 3-fold by LFP extract in MCF-7 cells
with apoptosis, as well as the cytoplasm of some morpholog-
ically healthy-looking cells; occasional nuclear staining was
Gene description UniGeneSymbol UniGeneID Fold
observed (Fig. 7). Caspase-3 labeling index (LI) in the tumor
increase
tissue of the treated mice was 29.45 ± 5.81% significantly
Cyclin-dependent kinase CDKN1A Hs.179665 3.00
higher than that (5.86 ± 2.20%) in the control mice (P < 0.05).
inhibitor 1A (p21, Cip1)
Superoxide dismutase 3, SOD3 Hs.2420 3.11
extracellular Discussion
Cytochrome P450, subfamily I CYP1A1 Hs.72912 7.04
(aromatic compound- Litchi fruit pericarp (LFP) contains significant amounts of
inducible), polypeptide 1
polyphenolic compounds, including condensed tannins (poly-
Insulin-like growth factor IGFBP6 Hs.274313 3.72
binding protein 6 meric proanthocyanidins), epicatechin, procyanidin A2, and
Alanyl-tRNA synthetase AARS Hs.75102 3.04 flavonoids (Sarni-Manchado et al., 2000; Zheng et al., 2003).
Sterol regulatory SREBF1 Hs.166 3.12 LFP extract has been shown to have antioxidant properties
element binding against fat oxidation in vitro (Zheng et al., 2003), but no
transcription factor 1
other bioactivity of LFP extract was reported. In vitro
ATP synthase, H+ ATP5JD Hs.64593 3.15
transporting, mitochondrial cytotoxicity assay, some plant extract exhibited potential
F1F0, subunit d antioxidant and anticancer properties (Primchanien et al.,
ADP-ribosyltransferase ADPRTL1 Hs.77225 29.25 2004; Ju et al., 2004) and inhibited proliferation of multiple
(NAD+; poly (ADP-ribose) human cancer cells (Magiatis et al., 2001; Kazi et al., 2003).
polymerase)-like 1
Animal studies have demonstrated that a dietary polyphenol
 X. Wang et al. / Toxicology and Applied Pharmacology 215 (2006) 168–178 173

Table 2 Table 2 (continued)

List of genes down-regulated more than 3-fold by LFP extract in MCF-7 cells
Gene description UniGeneSymbol UniGeneID Fold decrease
Gene description UniGeneSymbol UniGeneID Fold decrease
Protein kinase C binding PRKCBP1 Hs.75871 −4.26
Gardner–Rasheed feline FGR Hs.1422 −5.08 protein 1
sarcoma viral (v-fgr) Tousled-like kinase 2 TLK2 Hs.57553 −3.04
oncogene homolog Polo (Drosophila)-like kinase PLK Hs.77597 −3.74
Glucagon-like peptide GLP1R Hs.165 −5.14 Ribosomal protein S6 kinase, RPS6KA3 Hs.173965 −3.58
1 receptor 90 kDa, polypeptide 3
Protein phosphatase 2 PPP2CA Hs.91773 −3.10 Hyaluronan-mediated motility HMMR Hs.72550 −15.48
(formerly 2A), receptor (RHAMM)
catalytic subunit, A disintegrin and
alpha isoform metalloproteinase
Protein phosphatase 2 PPP2R1B Hs.108705 −3.18 domain 9 (meltrin gamma)
(formerly 2A), regulatory A disintegrin and ADAM9 Hs.2442 −5.09
subunit A (PR 65), metalloproteinase domain 9
beta isoform (meltrin gamma)
Nucleoside phosphorylase NP Hs.75514 −3.66 Tousled-like kinase 1 TLK1 Hs.18895 −3.37
Baculoviral IAP BIRC3 Hs.127799 −5.09 Cytochrome P450, CYP3A3 Hs.329704 −5.05
repeat-containing 3 subfamily IIIA
Interferon-induced IFI41 Hs.241510 −3.17 (niphedipine oxidase),
protein 41, 30 kDa polypeptide 3
Integrin, alpha 2 (CD49B, ITGA2 Hs.271986 −3.60 Splicing factor, arginine/ SFRS11 Hs.11482 −3.05
alpha 2 subunit of serine-rich 11
VLA-2 receptor) ATP-binding cassette, ABCE1 Hs.12013 −4.40
Transcription factor 12 TCF12 Hs.21704 −3.29 subfamily E
(HTF4, helix–loop–helix (OABP), member 1
transcription factors 4)
Interleukin 3 IL3 Hs.694 −6.46
(colony-stimulating known as tannic acid (TA) exhibits anticarcinogenic activity
factor, multiple) in chemically induced cancers (Gali-Muhtasib et al., 2001).
U4/U6-associated RNA HPRP3P Hs.11776 −5.28
Based on the potent antioxidant activity of LFP extract and its
splicing factor
Ste-20-related kinase SPAK Hs.199263 −3.10 major components of polyphenolic compounds and flavonoids
Budding uninhibited by BUB1 Hs.98658 −4.47 (Sarni-Manchado et al., 2000; Zheng et al., 2003), it could be
benzimidazoles 1 assumed that LFP extract might have anticancer activity
(yeast homolog) against some cancer cells and/or animal models. The present
Low density lipoprotein LRP5 Hs.6347 −3.49
study confirmed that LFP water-soluble CEE has strong dose-
receptor-related protein 5
Cyclin D binding DMTF1 Hs.5671 −4.74 and time-dependent anticancer activity against human breast
Myb-like transcription cancer cells (Fig. 1), with the IC50 at 80 μg/ml, and inhibited
factor 1 the colony growth potential of cancer cells in a dose-
Menage a trois 1 MNAT1 Hs.82380 −4.06 dependent manner (Fig. 2).
(CAK assembly factor)
Cultured cancer cells are valuable reagents for rapid
Potassium voltage-gated KCND1 Hs.55276 −3.29
channel, Shal-related screening of potential anticancer agents as well as for
subfamily, member 1 elucidation of mechanism of their activity. Prior to clinical
General transcription GTF2A2 Hs.76362 −6.35 trials, however, it is essential that the in vivo efficacy of
factor IIA, potential anticancer agents is determined in a suitable animal
2 (12 kDa subunit)
model (Singh et al., 2004). It was well known that human breast
Heat shock 105 kDa HSP105B Hs.36927 −3.37
TTK protein kinase TTK Hs.169840 −3.09 cancer cell line MCF-7 is ER positive, but approximately one-
Small inducible cytokine SCYA16 Hs.10458 −3.15 third of breast cancers are ER negative, carrying a worse
subfamily A (Cys–Cys), prognosis than the positive ones (Swami et al., 2003).
member 16 Therefore, it is important to discover new agents that are
Transcription factor CA150 CA150 Hs.13063 −3.05
effective on both ER positive and negative breast cancers. The
Tetracycline transporter- TETRAN Hs.157145 −4.49
like protein results of this experimental study demonstrated that LFP water-
KIAA0164 gene product KIAA0164 Hs.80338 −3.00 soluble CEE not only had powerful inhibitory effect on the
RNA binding motif, RBMS1 Hs.241567 −3.74 proliferation of both ER positive and ER negative human breast
single stranded cells in vitro but also significantly inhibited ER negative human
interacting protein 1
breast infiltrating duct carcinoma growth in vivo, without any
Replication factor C RFC1 Hs.166563 −3.82
(activator 1) 1 (145 kDa) untoward toxicity. These findings suggested that LFP water-
Splicing factor (CC1.3) CC1.3 Hs.145696 −5.69 soluble CEE might have potential anticancer activity on human
Interleukin 8 IL8 Hs.624 −3.54 breast cancer. There was no difference in antitumor activity of
LFP water-soluble CEE in either presence or absence of
estrogen, its action might be independent from estrogen-
174 X. Wang et al. / Toxicology and Applied Pharmacology 215 (2006) 168–178

Fig. 4. Oligonucleotide microarray analysis. Human breast cancer MCF-7 cells were treated with 100 μg/ml of LFP water-soluble CEE for 72 h, total RNA was
extracted from the treated and untreated cells, cRNA probes were created, and hybridized to HO4 ExpressChip, the hybridized slides were scanned with GMS418
Array Scanner. (A) Gene expression profiling of the untreated cells; (B) gene expression profiling of LFP water-soluble CEE-treated cells; (C) the histogram shows the
up-regulation and down-regulation of more than 5-fold.

regulatory mechanism, but further more experimental investi- The anticancer activity of LFP water-soluble CEE might
gation is needed to confirm its antitumor activity against ER result, at least in part, from inhibition of DNA synthesis,
positive breast cancer model in vivo. proliferation, as well as apoptosis induction of cancer cells.

Fig. 5. Comparison of microarray results with those determined by RT-PCR. (A) Gel image of 3 representative genes confirmed by RT-PCR. The amplified fragments
were quantified using β-actin for the normalization. (B) The correlation coefficient is shown in the graph.
 X. Wang et al. / Toxicology and Applied Pharmacology 215 (2006) 168–178 175

Fig. 6. Inhibition of tumor growth in nude mice xenograted with human breast IDC by LFP water-soluble CEE. Two weeks after orthotopical inoculation of human
breast IDC, the mice were randomly divided into two groups, six mice in control and seven mice in treatment group. In the treated mice, LFP water-soluble CEE
(0.3 mg/ml) was given through drink water ad libitum for 10 weeks. A significant reduction of tumor volume was observed in the treated mice. (A) Tumor bearing nude
mice of the untreated group (up row) and the treated group (down row); (B) tumor masses of the untreated group (up row) and the treated group (down row); (C) the
histogram shows that there was a significant difference of tumor volumes between the untreated and treated mice, the single asterisk (*) indicates a significant
difference from the control, one-way ANOVA, Tukey's test (P < 0.05).

Inhibition of DNA synthesis and proliferation of cancer cells CEE, BrdU-labeled cells in the treated cells reduced signifi-
were verified by its ability to reduce BrdU incorporation into cantly (P < 0.05), which indicated that LFP water-soluble CEE
cancer cells that correlates with decreased cell proliferation inhibited proliferation of cancer cells. Apoptosis induction was
(Milosevic et al., 2002) after treatment with LFP water-soluble additionally determined by increased caspase-3 protein

Fig. 7. Increase of caspase-3 protein expression by LFP water-soluble CEE. Caspase-3 protein expressions of tumor tissues derived from the untreated and treated mice
were detected through immunohistochemical staining. (A) Representative photographs of caspase-3 protein expression of the untreated mouse tumor; (B) the treated
mouse tumor; (C) the histogram shows that there was a significant increase of caspase-3 protein expression of LFP water-soluble CEE-treated mouse tumors, the single
asterisk (*) indicates a significant difference from the control, one-way ANOVA, Tukey's test (P < 0.05).
176 X. Wang et al. / Toxicology and Applied Pharmacology 215 (2006) 168–178
expression in tumors of the animals treated with LFP water-
soluble CEE.
The anticancer mechanism of LFP extract was further
investigated by oligonucleotide microarray; it was demonstrat-
ed that non-toxic dose of LFP water-soluble CEE affected the
gene expression profile of cancer cells by up-regulation of 41
genes (1.22%) and down-regulation of 129 genes (3.84%),
involved in various biological functions including cell cycle
regulation and cell proliferation, apoptosis, signal transduction
and transcriptional regulation, and extracellular matrix/adhesion
molecules, etc. The range of fold regulation varied widely with
ADPRTL1 exhibiting the maximum up-regulation (29.25-fold)
and RHAMM exhibiting the maximum down-regulation
(−15.48-fold).
The predominantly up- and down-regulated genes were
mainly associated with apoptosis, cell–cell and cell–matrix
interactions, motility, and invasiveness of cancer cells.
CYP1A1, encoding Cytochrome P450, subfamily I, up-
regulation is paralleled with cytotoxicity of aminoflavone in
sensitive breast tumor cells (Loaiza-Perez et al., 2004a, b),
resveratrol-induced differentiation and apoptosis of medullo-
blastoma cells (Liu et al., 2004), antiproliferative activity and
apoptosis induction of aminoflavone analogue (AF) of human
tumor renal cell carcinoma lines (Loaiza-Perez et al., 2004a, b),
and CYP1A1 activation led to cytotoxicity of anticancer agent
through DNA damage-induced apoptosis toxicity (Monks et al.,
2003). ADPRT gene encodes a zinc-finger DNA-binding
protein, poly (ADP-ribose) polymerase-1 (PARP-1), that
modifies various nuclear proteins by poly (ADP-ribosyl)ation
and functions as a key enzyme in the base excision repair
pathway; altered ADPRT/PARP-1 enzyme function was
associated with response to oxidative damage (Griesenbeck et
al., 1997; Lockett et al., 2004), during almost all forms of
apoptosis, PARP is activated by caspases (Hatip-Al-Khatib et
al., 2004), and inhibition of ADP-ribosyltransferase significant-
ly reduced its ability to induce apoptosis of cancer cells(Hatip-
Al-Khatib et al., 2004; Takamura-Enya et al., 2001). BIRC,
encoding apoptosis inhibitory protein was associated apoptosis
and cell cycles (Takita et al., 2004). Inhibitors of apoptosis
(IAPs) antagonize cell death and regulate the cell cycle (Dong et
al., 2002); decrease of c-IAP2 expression led to promotion of
apoptosis (Nishihara et al., 2003). In the present study, the
expressions of ADPRTL1 and CYP1A1 in LFP water-soluble
CEE-treated breast cancer cells increased and the expression of
BIRC3 decreased significantly; it could be inferred that LFP
water-soluble CEE inhibited proliferation and induced apopto-
sis of breast cancer cells mainly through up-regulation
expressions of CYP1A1 and ADPRTL1, and down-regulation
of BIRC genes.
For other predominantly down-regulated genes, ADAM
encoding a disintegrin and metalloprotease family contributes to
regulation of the cell–cell and cell–matrix interactions that are
critical determinants of malignancy; ADAM9 overexpression
enhances cell adhesion and invasion of non-small cell lung
cancer cells (Shintani et al., 2004), and is associated with poor
differentiation and shortened survival of pancreatic cancer
(Grutzmann et al., 2004). HMMR encoding the receptor for
hyaluronan mediated motility (RHAMM), a hyaluronan (HA)
binding protein, has been shown to play an important role in the
motility and invasiveness of malignant cells (Kuwabara et al.,
2004); expression and splicing of RHAMM are important
molecular determinants of disease severity in multiple myeloma
(Maxwell et al., 2004); and RHAMM blockade might be a
potential therapeutic target for difficult-to-treat neoplasm (Tolg
et al., 2003). The decreased expressions of ADAM9 (−5.09-
fold) and RHAMM (−15.48-fold) in the treated cancer cells
(Table 2, Fig. 4) suggested that LFP water-soluble CEE could
inhibit the motility and invasiveness of cancer cells, and
decreased their malignancy (Shintani et al., 2004; Grutzmann et
al., 2004; Kuwabara et al., 2004; Maxwell et al., 2004; Tolg et
al., 2003).
The most predominantly up-regulated gene after LFP water-
soluble CEE treatment was ADPRTL1 (29.25-fold) which was
associated with DNA repair, damage, and cell apoptosis
(Griesenbeck et al., 1997; Lockett et al., 2004; Hatip-Al-Khatib
et al., 2004; Takamura-Enya et al., 2001), and the most
predominantly down-regulated gene HMMR (−15.48-fold) was
associated with the motility and invasiveness of cancer cells.
The findings in this study showed LFP water-soluble CEE
resulting in DNA damage, proliferation inhibition and apoptosis
of cancer cells through up-regulation of ADPRTL1 expression,
and inhibition of the motility and invasiveness of cancer cells
through down-regulation of HMMR expression. ADPRTL1 and
HMMR might be the main drug target at which LFP water-
soluble CEE acted, but this needs to be confirmed in normal
and/or other cancer cells.
Although there was qualitative agreement between the
cDNA microassay and PCR assay, with 90% of genes tested
showing similar trend reported, false positive results were
common in cDNA microarray (Swami et al., 2003). Therefore,
3 representative genes in response to LFP water-soluble CEE
treatment were confirmed by semi-quantitative RT-PCR. A
good qualitative correlation was observed in the results obtained
by microarray and RT-PCR, but quantitative differences exist.
As cDNA microarray tended to have greater interassay variation
than quantitative RT-PCR (Swami et al., 2003), the existence of
quantitative differences is inevitable, even reverse correlation is
present (Swami et al., 2003; Morandi et al., 2006). The findings
in this work could show the trend of gene express profile of
cancer cells treated with LFP water-soluble CEE.
In conclusion, the potential anticancer activity of LFP extract
against human breast cancer was investigated in this experi-
mental study, for the first time. LFP extract exhibited a strong
inhibitory effect on the proliferation of both ER positive and
negative breast cancer cells in vitro and inhibited growth of ER
negative breast cancer in vivo. The results of this experimental
study suggested that Litchi fruit pericarp contains some
constituents, which would be useful for anticancer drug
discovery. The anticancer activity of LFP extract could be
attributed, in part to its proliferating inhibition and apoptosis
induction of cancer cells through up-regulation (CYP1A1,
ADPRTL1) and down-regulation (BIRC3, ADAM9, HMMR)
of multiple genes, which are involved in the cell cycle
regulation and cell proliferation, apoptosis, signal transduction
 X. Wang et al. / Toxicology and Applied Pharmacology 215 (2006) 168–178
and transcriptional regulation, motility, and invasiveness of
cancer cells. ADPRTL1, CYP1A1, and HMMR might be the
main molecular targets at which LFP water-soluble CEE acted.
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