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 Pdf
file on Apoptosis-inducing activity of the antimicrobial peptide
cecropin of Musca
domestica in
human hepatocellular carcinoma cell line BEL-7402 and the possible mechanism
Xiaobao Jin1,2,
Hanfang Mei1,2, Xiaobo Li1,2, Yan Ma1,2, Ai-hua
Zeng1,2, YanWang1,2, Xuemei Lu1,2, Fujiang
Chu1,2, Qiang Wu1,2, and Jiayong Zhu1,2*
1School of Basic Courses, 2Guangdong Key Laboratory of Pharmaceutical
Bioactive Substances, *Correspondence
address. Tel: +86-20-39352552; Fax: +86-20-39352222;
E-mail: [email protected]
We studied the apoptosis-inducing properties of the
antimicrobial peptide cecropin of Musca domestica in human
hepatocellular carcinoma cell line BEL-7402 and its underlying mechanism.
Proliferation inhibition of the human hepatocellular carcinoma BEL-7402 cells
and the human normal liver cells were determined by the MTT assay, and the
cell viability was determined by trypan blue dye exclusion assay. The
apoptotic tumor cells treated with cecropin were examined by transmission
electron microscopy and terminal-deoxynucleotidyl transferase mediated nick
end labeling. The apoptosis rate was measured by flow cytometry (FCM) with PI/Annexin-V
double staining. Western blot analysis and RT-PCR were used to determine the
expression levels of proteins involved in apoptosis, such as Fas, Fas-L,
caspase-8, and caspase-3. The experimental results showed that Musca domestica cecropin
inhibited the proliferation of human hepatocellular carcinoma BEL-7402 cells
in dose-dependent and time-dependent manners, without affecting the
proliferation of normal liver cells. FCM showed that the cell apoptosis rates
were 5.1±0.11%,
8.1± 0.04%, and 10.9±0.15% after the
treating with
Keywords
Musca domestica; antimicrobial peptide; cecropin; hepatocellular
carcinoma; apoptosis
Received:
November 2, 2009 Accepted: January 18, 2010
Introduction
The
housefly larvae have been used clinically to treat malnutritional stagnation,
decubital necrosis, osteomyelitis, ecthyma, and lip boil. They are also used
to treat coma and gastric cancer when combined with other drugs [1,2]. Recently, antitumor activities of the extract of
housefly larvae have been reported, but the antitumor mechanisms are still
unclear [2–5]. The chemical composition of M. domestica hemolymph is very complex, including antibacterial
proteins and carbohydrates, such as antimicrobial peptides, lysozyme, and
agglutinin [6,7]. There are increasing interests in the investigation of
the structures and functions of these active ingredients in the field of
entomology. We are interested in the antitumor activity of the extract of the M. domestica hemolymph, especially the antimicrobial peptides. To date,
only three antimicrobial peptides were isolated from M. domestica and they are cecropin, defensin, and attacin [8]. We have produced cecropin of M. domestica through the COS-7 eukaryotic expression system [9]. The aim
of the present research was to study the apoptosis-inducing activity of
cecropin in human hepatocellular carcinoma BEL-7402 cells and investigate the
underlying mechanism.
Materials and Methods
Preparation
of M. domestica antimicrobial peptide cecropin
Cecropin of
M. domestica was prepared through the COS-7 eukaryotic
expression system and purified to homogeneity by a nickel-chelating Sepharose
column as reported [9], with a purity of 99% identified by HPLC. It
has the amino acid sequence of
MNFNKLFVFVALVLAVCIGQSEAGWLKKIGKKIERVGQHTRDATIQTIGVAQQAANVAATLKG. Before use,
the peptide was dissolved in RPMI 1640 medium at a concentration of
Cell
lines
The human
hepatocellular carcinoma cell line BEL-7402 and Chang normal liver cell line
(The Cell Bank of Type Culture Collection of Chinese Academy of Sciences,
Shanghai, China) were maintained in RPMI 1640 medium with 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin under 5% CO2 at 37ºC in a humidified incubator.
Design
and synthesis of primers
Fas, Fas-L, caspase-8, caspase-3, and b-actin gene specific primers were designed by software primer
premier 5.0, which were synthesized by Shanghai Biotechnology Co. Ltd
(Shanghai, China). All primers are listed in Table 1.
Cell
proliferation assay
Human
hepatocellular carcinoma BEL-7402 cells and Chang normal liver cells were
plated into the 96-well tissue culture plates (5.0 ´ 103 cells per well), then incubated at 37ºC overnight. The next day, the media were
replaced with 200 ml of fresh complete medium containing cecropin of different final
concentrations, and no cecropin was added to the control well. After 24, 48,
or 72 h, the supernatants were removed and cell layers were washed with PBS
and incubated with MTT (50 ml, 0.5 mg/ml) in RPMI 1640
without FBS for 4 h at 37ºC. The cell cultures were centrifuged at
Cell
viability determined by trypan blue dye exclusion assay
For the
trypan blue dye exclusion assay, 5.0 ´ 103 cells were seeded into 24-well plates and
treated with or without (as control) cecropin at specified doses for 24, 48,
or 72 h. Both floating and adherent cells were collected and stained by trypan
blue. The stained cells were microscopically counted at five random high-power
fields and the number of dead cells was counted and expressed as a percentage
of the total number of cells counted.
Transmission
electron microscopy
Totally, 3 ´ 104 BEL-7402 cells in 2 ml RPMI 1640 were seeded into 6-well
plates at 37ºC overnight. The next day,
cecropin was added to a final concentration of
Terminal
deoxynucleotidyl transferase-mediated nick-end labeling
Apoptotic
cells were detected by terminal deoxynucleotidyl transferase-mediated nick-end
labeling (TUNEL) labeling detection of free 3‘-OH groups in fragmented DNA in situ using ApopTagw peroxidase in situ apoptosis detection kit (Chemicon, Temecula,
USA). About 3 ´ 105 BEL-7402 cells were seeded into 6-well plates and treated
with or without (as control) specified doses of cecropin for 72 h, fixed in 4%
paraformaldehyde in PBS for 1 h at room temperature, and permeabilized in PBS
containing 0.1% Triton X-100 and 0.1% sodium citrate for 2 min on ice. After
nick-end labeling with digoxigenin-deoxyuridine triphosphate by terminal
deoxynucleotidyl transferase, immunostaining was performed using peroxidase-conjugated
antidigoxigenin antibody. Apoptotic cells were visualized with
diaminobenzidine substrate, becoming a dark-brown color. Specimens were then
counterstained with hematoxylin. Images were captured using a microscope
attached to a charge-coupled device (CCD) camera.
Apoptosis
rate determined by flow cytometry
BEL-7402
cells at 5.0 ´ 105 cells/ml were inoculated into 6-well culture plate and
incubated at 37ºC. The next day, after the
medium was removed, 2.0 ml of RPMI 1640 complete medium with the final
concentrations of cecropin at 25, 50, and
Western
blot analysis
Cultures of
BEL-7402 cells at approximately 80% confluence were treated with
RT-PCR
detection of Fas, Fas-L,
caspase-8, caspase-3 expression
Cultures of
BEL-7402 cells at approximately 80% confluence were treated with
Statistical
analysis
The data
were presented as the mean±SD. Comparisons of the data were analyzed by SPSS 11.0 using one-way ANOVA
LSD statistical analysis method. P< 0.05 was
considered statistically significant.
Results
Cell
proliferation and cell viability assay
MTT results
showed that M.
domestica cecropin inhibited the
proliferation of BEL-7402 cells within 12.5–Trypan blue
dye exclusion assay revealed that M. domestica cecropin decreased cell viability of BEL-7402 cells in the
concentration range of 12.5–
Transmission
electron microscopy and TUNEL assay
The
morphology of control BEL-7402 cells at
Fas, Fas-L,
caspase-8, and caspase-3 gene expression
RT-PCR
results showed that the Fas, Fas-L, caspase-8, and caspase-3
gene expression levels of
BEL-7402 cells were increased after the treating with M. domestica cecropin (Fig. 4). The Fas, Fas-L, and caspase-8 mRNA levels increased after 12 h treatment of M. domestica cecropin, whereas caspase-3 mRNA level of BEL-7402 cells increased after 24 h
treatment of M.
domestica cecropin.
Discussion
Insect
cecropin is a class of antimicrobial peptides that were first isolated by the
Boman et al. [10,11] in Hyatophora cecropia pupae. Later on, a number of similar types of cecropin
antimicrobial peptides were isolated. To date, there are more than 20 known
cecropin peptides. Studies have found that cecropins inhibited the growth of
Gram-positive bacteria, Gram-negative bacteria, fungi, viruses, and certain
parasites. Furthermore, recent researches have found that insect cecropins can
inhibit the proliferation of certain types of tumor cells, but they did not
affect the proliferation of normal cells, and found membrane differences
between the cell membranes of tumor cells and normal cells that contribute to
the selectivity of insect cecropins for tumor cells [12–14]. Because of their selectivity, this type of peptides
would be a good candidate for the development of antitumor agents. In recent
years, a number of studies have reported that the extract of M. domestica hemolymph can inhibit the growth of tumor cells [2–5]. Whether the antibacterial peptides play a role in the
antitumor activity is unknown. We cloned the housefly cecropin gene (GenBank
accession no. EF175878) and showed that the full-length ORF contains 192 bp
and encodes a 63-amino acid peptide [15].
But the
housefly cecropin does not contain the conserved alanine-glycine-proline (Ala-Gly-Pro)
segment of typical insect antibacterial peptides. Musca domestica cecropin showed 85 and 84% homology with the Sarcophaga peregrina sacrotoxin IB and the Mediterranean fruit fly cecropin,
respectively [15]. These results show that the peptide
structure of M.
domestica cecropin is basically similar to
that of insect cecropins, but some unique structural features remain. In this
paper, we examined the antitumor activity of M. domestica cecropin and found that it inhibited the proliferation of
the human hepatoma BEL-7402 cells in dose- and time-dependent manners. It did
not affect the proliferation of normal liver cells, confirming its selectivity
towards cancer cells. The tumor cell IR reached 65.7% after 72 h treatment
with
To
understand the tumor cell apoptosis mechanism of cecropin, we examined its
effects on the expression of proteins that are involved in apoptosis. It is
currently known that apoptosis is mediated by two major pathways [17,18]. One is the Fas/Fas-L and TNF/TNFR system, also called the death
receptor-mediated signal transduction pathway. The other one is the endogenous
mitochondria signal transduction pathway. Fas, also called CD95 or Apol, is a
cell surface receptor, belonging to the tumor necrosis factor family. Once
activated by binding to its ligand Fas-L, Fas is translocated into the
cytoplasm and formed the so-called death-induced signal transduction complex (DISO).
DISO causes the activation of caspase-8, which mediates apoptosis. Our western
blotting and RT-PCR results showed that the expression levels of Fas/Fas-L and
caspase-8 increased after treatment with cecropin for 12 h, and that of
caspase-3 increased after treatment for 24 h. In addition, our results showed
that the expression of Bax/Bcl-2 and release of cytochrome C were not
influenced before and after cecropin treatment (data not shown).
In
conclusion, this study shows that housefly cecropin possesses antibacterial
activity and has in
vitro antitumor activity. But it does
not show any inhibitory effect on the proliferation of normal liver cells. Its
specificity towards cancer cells makes it a good candidate for further
investigation as an antitumor agent. We also found that housefly cecropin can
induce apoptosis of the human hepatoma BEL-7402 cells, which might be
associated with up-regulation of Fas, Fas-L, caspase-8, and caspase-3. This
shows that the antimicrobial peptide cecropin-induced apoptosis in human
hepatocellular carcinoma BEL-7402 cells probably by triggering extrinsic
apoptotic pathway.
Funding
This work
was supported by grants from the National Natural Science Foundation of China
(no. 30671832), the Key Science and Technology Foundation of Guangdong
Province (no. 2003B31602), and the Key Science and Technology Foundation of
Guangzhou (no. 2005Z3-E0211).
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