Pro-apoptotic effect of cecropin AD on nasopharyngeal carcinoma cells

Nasopharyngeal 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 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 ofCadmRNA 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 showed that the recombinant plasmid pSectagcad contained the integral Cad gene and right reading box. Therefore, this recombinant plasmid pSectagcad was 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.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.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 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 condensation and margination. But the cells in control group were intact and nearly invisible. NIH3T3-pSec-tagcad cells had no significant change compared with the NIH3T3-pSectag2hygroA cells (Fig. 2). These results indicated that Cad gene had no effect on the normal eukaryotic cell.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.DNA ladder analysis Compared with the control, the CNE2 cells transfected with pSectagcad showed distinct DNA apoptosis ladder from the agarose gel electrophoresis results (Fig. 3). This further confirmed that Cad could induce the CNE2 cell apoptosis.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.Cell apoptosis by flow cytometric analysis The rates of apoptotic cells measured by flow cytometric analysis after 48 hours in the CNE2-pSectagcad group and in the CNE2-pSectag2hygroA group were (14.575 ± 0.892)%, (2.800 ± 0.535)% (n=5 in each group), respectively. 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 and swelling, 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 natural cecropins.9 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 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 such antimicrobial 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.

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