Pro-apoptotic effect of cecropin AD on nasopharyngeal carcinoma cells.

Full text

Page 1

Chin Med J 2006; 119(12):1042-1046
1042
Brief report

Pro-apoptotic effect of cecropin AD on nasopharyngeal
carcinoma cells

XIAO Ye-chen, HUANG Ya-dong, XU Pei-lin, ZHOU Zhen-qing and LI Xiao-kun

Keywords: cecropin AD; nasopharyngeal carcinoma; CNE2 cells; apoptosis

asopharyngeal carcinoma (NPC) is one of the
highly prevalent malignancies in Southeast
Asia.1 Chemotherapy and radiotherapy are
commonly administrated to treat nasopharyngeal
carcinoma, but these therapies can not prevent the
recurrence and metastasis of tumor cells.2
Furthermore, these therapies have severe side
effects. Therefore, it is important to find a more
effective therapy, e. g., a gene therapy for treatment
of nasopharyngeal carcinoma. Cecropin AD (Cad) is
a hybrid peptide that exhibits higher antibacterial
activity than natural cecropin. It consists of 1－11
amino acid residues in the N-end of cecropin A and
12－37 amino acid residues in the c-end of cecropin
D.3 The role of Cad in the malignant cells remains
unclear. In this study, the Cad gene was cloned and
transferred into NPC CNE2 cells and its
pro-apoptotic effect were investigated.

METHODS

Construction of recombinant expression vector
Two primers: P1 (5'-GCGGATCCAATGAAGTGGAA
GTTGTTCAA-3' carrying a BamH I site, underlined)
and P2 (5'-GCCTCGAGTTATTACTTAGCCAAGG
CAG-3', carrying an Xho I site, underlined) were
designed for the Cad gene. The Cad gene was
amplified by polymerase chain reaction (PCR) with
primer P1 and P2 from 1 ng pPICZcad. PCR
condition: denaturing at 95˚C for 30 seconds,
annealing at 55˚C for 30 seconds, and extension at
72˚C for 30 seconds by 30 cycles. Final extension
was performed at 72˚C for 10 minutes. The fragment
carrying BamH I and Xho I sites was acquired by
PCR, size of which was about 142 bp. The product
was doubly digested by BamH I and Xho I enzymes,
and then ligated to the 5.7 kb fragment of
pSectag2hygroA (Invitrogen, USA) digested with the
same enzymes. The ligated product was
transformed into E. coli DH5α. Restriction
endonuclease analysis, PCR and sequencing were
performed to validate the reading box of the
recombinant plasmid.

Cell culture and transfection
The CNE2 cells were cultured in RPMI 1640 medium
supplemented with 10% calf bovine serum, 100 U/ml
penicillin, 100 U/ml streptomycin and humidified 5%
CO2 at 37˚C. The NIH3T3 cells that adopted the
same culture condition were used as control of
natural eukaryotic cells. Twelve hours before
transfection, cells were seeded onto 24-well plates at
a density of 5×104 cells per well. The confluence
would reach approximately 70%－80% at the time of
transfection. Cells were transfected with 0.25 µg/well
of pSectagcad vector using 1 µl Avantgen reagent
(Allele, USA) following the protocol provided by the
manufacturer. Forty-eight hours later, the cells were
harvested for the following experiments.

Gene expression
Total RNA was isolated using the RNAex reagent
(Bocai company, China) according to the protocol.
The RNA concentration and purity were assessed
with optical density measurements. Reverse
transcription PCR (RT-PCR) was performed to
detect Cad mRNA expression in transfected CNE2

Biotechnology Research Center, Key Laboratory of Gene
Engineering, Ministry of Education, Sun Yat-sen University,
Guangzhou 510275, China (Xiao YC and Xu PL)
Biopharmaceutical Research & Development Center, Jinan
University, Guangzhou 510632, China (Huang YD)
Department of Zoology, Miami University, Oxford 45056, USA
(Zhou ZQ)
School of Pharmaceutical Sciences, Wenzhou Medical College,
Wenzhou 325035, China (Li XK)
Correspondence to: Dr. LI Xiao-kun, School of Pharmaceutical
Sciences, Wenzhou Medical College, Wenzhou 325035, China (Tel:
86-431-4533327. Email: xiaokunli@163.net)
This work was supported by a grant from the National 973 Project
of China (No. G1999054204).
N

Page 2

Chinese Medical Journal 2006; 119(12):1042-1046
1043
cells. Reverse transcription of 2 mg of total RNA was
performed using 20 U of AMV reverse transcriptase
(BBI, Canada), 0.5 mg of oligo(dT)12-18 primer, 0.5
mmol/L each of dNTP and 20 U of RNase inhibitor in
a total volume of 20 µl at 42˚C for 60 minutes. The
reaction was terminated by heating the mixture at
70˚C for 10 minutes.

Cell proliferation assay
Cells were seeded at 7×103 cells per well on 96-well
plate and cultured in the presence of 10% fetal
bovine serum for one day. After 48 hours, the cells
transfected with pSectagcad or pSectag2/hygroA
were harvested.

During the last 4 hours of each treatment, cells were
pulsed with MTT [3-(4,5-dimethylthiazolyl-2)-2, 5-
diphenyltetrazolium bromide, Sigma, USA] 10 µl/well
[5 mg/ml in phosphate-buffered saline (PBS)] to
measure the cell growth. The purple-blue MTT
formazan precipitate was dissolved in 110 µl of
DMSO and swirled 30 minutes. Absorbance was
measured with spectrophotometer at the wavelength
of 570 nm. Five repeat elements were conducted on
each treatment.

Morphological changes
The cells transfected with pSectagcad or pSectag2-
hygroA were incubated in RPMI-1640 medium for 48
hours. They were fixed with methanol and air-dried
after washing twice with PBS, and stained with
May-Grunwald-Giemsa (Sigma, USA) for 10 minutes.
Dyestuff was discarded and cells were washed.
Then the cell morphology was observed under a
microscope.

DNA fragmentation assay
The cells transfected with pSectagcad or pSectag2-
hygroA were cultured in RPMI-1640 medium for 48
hours and then collected for genomic DNA extraction.
Approximately 10 µg DNA was loaded on 1.5%
agarose gel for electrophoresis.

Apoptosis assay by flow cytometry
Cell apoptosis was analyzed by flow cytometry.
Around 1×106 cells were collected and washed with
PBS and then fixed with ice-cold 70% ethanol and
stored at 4˚C for following use. After being
resuspended in PBS containing 100 g/ml RNase A
and 20 g/ml propidiumodide (PI) for 30 minutes,
samples were analyzed by flow cytometry (FACS
Calibur, BD, USA). The apoptotic cells were
observed as a subdiploid peak.

Statistical analysis
The data are expressed as the mean±standard
deviation (SD) and analyzed by SPSS 10.0 software.
Independent-samples t test was used and P<0.05
were considered statistically significant.

RESULTS

Recombinant plasmid construction and
expression of Cad mRNA
The Cad gene contains BamH I and Xho I enzyme
digestion sites and was amplified from the pPICZcad
plasmid which was about 142 bp. The recombinant
plasmid was confirmed by enzyme digestion and
sequencing. The results
recombinant plasmid pSectagcad contained the
integral Cad gene and right reading box. Therefore,
this recombinant plasmid
constructed successfully.

The expression of Cad mRNA was detected by
RT-PCR. The results showed Cad mRNA in the
transfected cells was overexpressed compared with
that in the untransfected cells (Fig. 1). Beta-actin
was used as internal control.

Suppression of cell growth
The absorbance values (570 nm) of CNE2 cells,
CNE2-pSectag2hygroA cells and CNE2-pSectagcad
cells were 0.541±0.011, 0.521±0.007, 0.459±
0.010 (n=5 in each group), respectively. There were
statistically significant differences between CNE2-
pSectag2-hygroA cells and CNE2-pSectagcad cells
(F=1.120, t=5.757, P<0.01). These results indicated
that the Cad protein expressed in the CNE2 cells
strongly inhibited cell proliferation.

The absorbance values (570 nm) of NIH3T3 cells,
NIH3T3-pSectag2hygroA cells and NIH3T3-pSec-
tagcad cells were 0.561±0.004, 0.552±0.010,
0.545±0.006 (n=5 in each group), respectively. The
growth of NIH3T3 cells expressing the Cad gene had
no significant difference compared with the
NIH3T3-pSectag2hygroA cells (F=0.469, t=1.28,
P>0.05). Hence, the Cad gene had no inhibitory
effect on the natural eukaryotic cells.

Morphological detection
The distinct apoptosis characters in morphological
showed that the
pSectagcad was

Page 3

Chin Med J 2006; 119(12):1042-1046
1044


Fig. 1. Cad gene was transfected into the CNE2 and
NIH3T3 cells, and its mRNA level was detected by
RT-PCR. Lane A: CNE2-pSectag2hygroA plasmid; Lane
B: NIH3T3-pSectag2hygroA plasmid; Lane C: NIH3T3-
pSectagcad plasmid; Lane D: CNE2-pSectagcad
plasmid; M: marker. The Cad mRNA level was higher in
the transfected cells (C, D) than that in untransfected
cells (A, B). Beta-actin was used as internal control.

changes were detected in the transfected CNE2 cell
lines treated with Giemsa (Fig. 2). The small
apoptosis bodies appeared near the cells, showing
cell shrinkage, chromatin
margination. But the cells in control group were intact
and nearly invisible. NIH3T3-pSec-tagcad cells had
no significant change
NIH3T3-pSectag2hygroA cells (Fig. 2). These results
indicated that Cad gene had no effect on the normal
eukaryotic cell.

DNA ladder analysis
Compared with the control, the CNE2 cells
transfected with pSectagcad showed distinct DNA
apoptosis ladder from
electrophoresis results (Fig. 3). This further
confirmed that Cad could induce the CNE2 cell
apoptosis.

Cell apoptosis by flow cytometric analysis
The rates of apoptotic cells measured by flow
cytometric analysis after
CNE2-pSectagcad group and in the CNE2-
pSectag2hygroA group were (14.575 ± 0.892)%,
(2.800±0.535)% (n=5 in each group), respectively.
condensation and
compared with the
the agarose gel
48 hours in the



Fig. 2. Morphological changes of the apoptotic cells (Giemsa). The transfected CNE2 cells were treated with Giemsa. A and
B: CNE2 cells and CNE2-pSectag2hygroA cells, respectively, were intact and nearly invisible. C: CNE2-pSectagcad cells
showed cell shrinkage, chromatin condensation and margination. D and E and F: morphologies of NIH3T3 cells,
NIH3T3-pSectag2hygroA cells and NIH3T3-pSectagcad cells, respectively, showed no significant difference. Bar=20 µm.

Page 4

Chinese Medical Journal 2006; 119(12):1042-1046
1045


Fig. 3. DNA ladder of the apoptotic cells. Lane A:
CNE2-pSectag2hygroA cells showed no characteristic
apoptotic ladder-like pattern; lanes B and C: CNE2-
pSectagcad cells showed characteristic apoptotic
ladder-like pattern.

The rate of apoptotic cells in the test group was
significantly increased compared with that in the
control group (F=0.561, t=21.96, P<0.01).

DISCUSSION

The mechanism by which antibacterial peptides,
such as Cecropins, exert their anti-tumor effect
remains unknown. However, these peptides were
believed to exert their anticancer effect by interacting
strongly with the anionic phospholipids on the cell
surface of the tumor cells,4,5 or by triggering
mitochondrial permeabilization
resulting in release of cytochrome c and induction of
apoptosis,6,7 or by forming ion channels/pores.8

Cecropin AD is a hybrid peptide that exhibits higher
antibacterial activity than
However, it is not known whether Cad gene is
against the cancer cells. We cloned the Cad gene
and constructed eukaryotic expression vector
pSectagcad. The Cecropin gene was inserted
downstream of the transcriptional control of Human
cytomegalovirus (CMV) immediate-early promoter/
enhancer. We could only detect the expression of
Cad gene at RNA level. In the future, western
blotting should be performed to further confirm the
expression of Cad gene at protein level. Cad was
voltage-dependent pore forming peptide. This
structural character made it insensitive to eukaryotic
cells.9 The results showed that the growth of NIH3T3
cells was not affected
and swelling,
natural cecropins.9
by the transfected
recombinant plasmid and no apoptotic characters
had been detected morphologically. This further
proved that the antibacterial peptide Cad had no
effect on normal eukaryotic cell and Cad gene
significantly inhibited the growth of CNE2 cells and
induced their apoptosis. These studies suggested
that the antimicrobial peptides had gene therapy
potential for cancer treatment. Cecropins have been
reported to have destructive effects on cytoskeleton
of human nasopharyngeal carcinoma cell lines
CNE2.10 The structure of Cad is similar to that of
Cecropins. Presumably, Cad can result in apoptosis
by destroying the cytoskeleton of CNE2 cells.

Summarily, antibacterial peptide Cad was able to
express in the eukaryotic cells. Its transient
expression significantly suppressed the growth of
tumor cells and induced cells apoptosis. But it had
no effect on the general eukaryotic cells. Most
importantly, our data suggested the possibility of
delivering and expressing
peptides to tumor cells in vivo. In the future, more
work should be done on screening the stable
expression of cell lines, performing experiments on
animals, and practical clinical research.

REFERENCES

1. McDermott AL, Dutt SN, Watkinson JC. The aetiology of
nasopharyngeal carcinoma. Clin Otolaryngol 2001; 26:
82-92.
2. Freytag SO, Kim JH, Brown SL, Barton K, Lu M, Chung
M. Gene therapy strategies to improve the effectiveness
of cancer radiotherapy. Expert Opin Biol Ther 2004; 4:
1757-1770.
3. Stelner HD, Hultmark A, Engstrdom H, Bennich H,
Boman HG. Sequence and specificity of two antibacterial
proteins involved in insect immunity. Nature 1981; 292:
246-248.
4. Shin SY, Kang JH, Jang SY, Kim SD, Ryu PD, Hahm KS,
et al. Effects of the hinge region of cecropin A
(1-8)-magainin 2(1-12), a synthetic antimicrobial peptide,
on liposomes, bacterial and tumor cells. Biochim Biophys
Acta 2000; 1463: 209-218.
5. Utsugi T, Schroit AJ, Connor J, Bucana CD, Fidler IJ.
Elevated expression of phosphatidylserine in the outer
membrane leaflet of human tumor cells and recognition
by activated human blood monocytes. Cancer Res 1991;
51: 3062-3066.
6. Mai JC, Mi Z, Kim SH, Ng B, Robbins PD. A proapoptotic
such antimicrobial

Page 5

Chin Med J 2006; 119(12):1042-1046
1046
peptide for the treatment of solid tumors. Cancer Res
2001; 61: 7709-7712.
7. Risso A, Braidot E, Sordano MC, VianelloA, Macri F,
Skerlavaj B. BMAP-28, an antibiotic peptide of innate
immunity, induces cell death through opening of the
mitochondrial permeability transition pore. Mol Cell Biol
2002; 22: 1926-1935.
8. Cruciani RA, Barker JL, Zasloff M, Chen HC, Colamonici
O. Antibiotic magainins exert cytolytic activity against
transformed cell lines through channel formation. Proc
Natl Acad Sci U S A 1991; 88: 3792-3796.
9. Christensen B, Fink J, Merrifield RB, Mauzerall D.


Channel-forming properties of cecropins and related
model compounds incorporated into planar lipid
membranes. Proc Natl Acad Sci U S A 1988; 85:
5072-5076.
10. Ling BX, Meng QL, Wang JN, Bao WH, Wu J. Effect of
cecropin on cytoskeleton of human nasopharyngeal
carcinoma cell line CNE2. J Nanjing Mil Med Coll (Chin)
2003; 25: 1-3.

(Received April 19, 2006)
Edited by GUO Li-shao