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Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 217-225 |
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doi:10.1111/j.1745-7270.2008.00389.x |
Identification and
characterization of a novel peptide ligand of Tie2 for targeting gene therapy
Xianghua Wu1,
Zonghai Li2, Ming Yao2, Huamao Wang2, Sumin Qu2,
Xianlian Chen2, Jinjun Li2, Ye Sun3, Yuhong Xu2,3,
and Jianren Gu2*
1 Department of Medical Oncology, Cancer Hospital
of Fudan University, Shanghai 200032, China
2 National Laboratory for Oncogenes and Related
Genes, Shanghai Cancer
Institute, Shanghai
Jiao-Tong University Medical School, Shanghai 200032, China
3 School of Pharmacy, Shanghai Jiao-Tong University,
Shanghai 200030, China
Received: September 4, 2007�������
Accepted: December
12, 2007
This work was
supported by a grant from the Major State Basic Research Development Program of
China (No. 2004CB518802)
*Corresponding author:
Tel/Fax, 86-21-64177401; E-mail, [email protected]
Tyrosine
kinase with immunoglobulin and epidermal growth factor homology domain-2 (Tie2)
has been considered as a rational target for gene therapy in solid tumors. In
order to identify a novel peptide ligand of Tie2 for targeted gene therapy, we
screened a phage display peptide library and identified a candidate peptide
ligand NSLSNASEFRAPY (designated GA5). Binding assays and Scatchard analysis
revealed that GA5 could specifically bind to Tie2 with a dissociation constant
of 2.1�10-8 M. In
addition, we showed that GA5 was internalized into tumor cells highly
expressing Tie2. In the biodistribution assay, 125I-GA5 was
mainly accumulated in SPC-A1 xenograft tumors that express Tie2. In gene
delivery studies, GA5-conjugated polyethylenimine vector could achieve greater
transgene transduction than non-targeted vectors both in vitro and in
vivo. Tumor growth inhibition was observed in SPC-A1 xenograft-bearing mice
that received eight intratumoral injections of GA5-polyethylenimine/p53
complexes in 3 weeks. The difference in tumor volume between the experiment and
control groups was significant (p<0.05).
Our results showed that GA5 is a potentially efficient targeting element for
cancer gene or molecular therapy.
Keywords������� Tie2;
gene therapy; phage display; polyethylenimine; p53 gene
Targeted therapeutic gene delivery into a desired tumor cell or tissue is of unquestionable importance for improving therapeutic efficacy and minimizing adverse effects derived from random distribution of therapeutic agents [1,2]. However, gene therapy is currently limited by the difficulty of achieving efficient gene delivery into defined target cells. To overcome the common disadvantages of current gene delivery vehicles, many efforts have been made to design optimal vehicles for efficient gene delivery [3-5]. A receptor-mediated gene delivery system has been developed as an attractive approach that can potentially target genes into defined cells over-expressing cellular membrane receptor. It is particularly interesting because of its potential to circumvent the main disadvantage of viral vector [6]. Polyethylenimine (PEI) derivatives are linear (22 kda) or branched (25 kda) molecules that were shown to be efficient in gene transfer in vitro and in vivo [7,8]. They can target genes into desired cells successfully based on recognizable receptor-mediated gene delivery system. PEI/DNA complexes conjugated with the cell-binding epidermal growth factor receptor (EGFR) peptide ligand could achieve 10-fold to 100-fold higher gene expression levels in tumor tissue than in other tissues [9]. Recently, small molecular peptide ligands have been pursued as potential tumor targeting agents for selective delivery of therapeutic genes to tumors [10-13]. When compared with macromolecular natural ligands, these small-sized peptides have the advantages of readily diffusible ability, less immunogenicity, and higher target-to-background ratios [14,15]. Tyrosine kinase with immunoglobulin and epidermal growth factor homology domain-2 (Tie2) has been described as playing a critical role in the angiogenesis process in various cancers and becomes a novel marker of microvasculature of solid tumors [16-18]. Targeting Tie2 in gene therapy has been proposed as a potentially powerful approach for the treatment of cancer [19-21].
Tumor suppressor gene, p53, was found to be mutated in many solid tumors [22]. In lung cancer, mutated p53 was found in nearly 50% of cases and was associated with poor prognosis [23]. Because p53 and PI3K/Akt regulate cell survival and death [24], different approaches for targeting p53 replacement gene therapies have been explored [25-28]. PEI/wt p53 transfection can inhibit the growth of human head and neck squamous cell carcinoma xenografts with mutated p53 in mice [29].
In this study, we identified a peptide ligand of Tie2 by screening a phage display peptide library and investigated its targeting effect in vitro and in vivo. We also conjugated the peptide ligand with PEI to construct a Tie2-mediated non-viral gene delivery vector and used the vector to transfer the reporter gene in vitro and in vivo. The targeted gene therapy experiment was carried out in mice bearing human lung carcinoma xenografts by iteratively transferring the wild-type p53 gene with the gene vector.
Materials and methods
Cell culture and xenograft
tumor model
SMMC7721, a human hepatic carcinoma cell line that negatively expresses Tie2 [30], was a gift from the second university of military medicine (Shanghai, China). SMMC7721-ExTie2 was derived from SMMC7721 cells transfected with the plasmid pcDNA3.0-ExTie2 (a plasmid constructed by inserting a cDNA fragment of the extracellular and transmembrane domain of Tie2 into EcoRI/XhoI sites of plasmid pcDNA3.0) and selected by G418 (Stratagene, La Jolla, USA) [30]. SPC-A1, a human lung adenocarcinoma cell line expressing Tie2 [30], was provided by American Type Culture Collection (Manassas, USA) and was used to establish the xenograft tumor models for in vivo assay. These cells were incubated at 37 �C in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (Gibco BRL, Carlsbad, USA) in a humidified 5% CO2 atmosphere.
Biopanning
The Ph.D-12 phage display peptide library was purchased from New
England Biolabs (Beverly, USA). The procedure for screening the phage display
library was modified according to the manufacturer's instructions. Briefly, cultured
SMMC7721-ExTie2 cells were washed with phosphate-buffered saline (PBS), then
the phage library (4�1010 pfu/per well) diluted in 1 ml of DMEM containing 1% (w/v) bovine serum albumin (BSA; Sigma-Aldrich, St. Louis,
USA) was added per well. Phages were allowed to incubate with cells for 1 h at
37 �C. Unbound phages were removed by washing 10 times with Tris-based buffered
saline containing 0.1% (v/v) tween-20 (TBST). Finally, phages bound to Tie2 receptors were
eluted specifically by adding TBS containing 10 mg/ml angiopoietin-2
(R&D Systems, Minneapolis, USA) to each well with gentle agitation for 1 h
at 37 �C. Eluted phages were titered and amplified in Escherichia coli ER2537
cells then reapplied in subsequent rounds of panning. The elution procedure was
repeated four times and the final elute was used for amplification and
titration. Individual
blue clones were randomly selected and amplified by infecting ER2537 cells.
Phage single-strand DNA was isolated for sequencing.
The candidate peptide sequence was determined by amino acid sequence analysis
displayed on the most enriched phages.
�
Binding activity evaluation of
enriched phages
SMMC7721-ExTie2 cells, SPC-A1 cells, and SMMC7721 cells were seeded
in 96-well plates at a density of 1�104
cells per well. After blocking with PBS containing 1% BSA, 1�1010 pfu phages were added to each well and incubated
with the cells for 60 min at 37 �C, then washed 10 times with cold TBST. Bound phages were detected by incubation with a 1:5000 horseradish peroxidase-conjugated
anti-M13 antibody (Amersham Biosciences, Piscataway, USA) for 1 h, followed by
washing and the addition of a peroxidase substrate (o-phenylenediamine,
0.4 mg/ml) in citrate-phosphate buffer (pH 5.0) containing 0.02% (v/v)
H2O2. The reaction was stopped with 50 ml of 12.5% H2SO4. A405 was
determined by using a Bio-Rad model 550 microplate reader (Bio-Rad, Hercules,
USA). In the phage recovery assay, recombined human Tie2/Fc protein (rh-Tie2;
R&D Systems) and BSA were immobilized on 96-well plates with 50 ml NaHCO3 (0.1 M, pH 8.6) per well overnight at 4 �C. A total of 2�1010 pfu enriched and insertless phages diluted into 50 ml TBS containing
1% BSA were added to each well. After incubation for 1 h at 37 �C under gentle
agitation, unbound phages were washed 10 times with TBST. Phages
bound to Tie2 receptors were eluted with 100 ml glycine-HCl (0.2 M, pH 2.2) for 10 min at room temperature. Recovery phages were determined
by titrating on X-gal/IPTG agar plates [7].
Peptide synthesis and binding
assay
Candidate peptide NSLSNASEFRAPY (designated GA5) and an irrelevant peptide, HY12 (HATGTHGLSLSHY), were synthesized (GL Biochem, shanghai, China). The chloramine-T procedure was used to radioiodinate GA5 and HY12 with 125I [31]. For the in vitro binding assay, 1�104 SMMC7721-ExTie2 or SMMC7721 cells per well were inoculated in 96-well plates, and grown until cells reached 70% confluence. Cells were then washed three times with PBS, then 1�105 cpm (counts per minute) 125I-GA5 was added into 100 ml binding buffer [DMEM containing 0.1% (W/V) BSA]. After incubating at 37 �C for 30 min, cells were washed three times with PBST to remove unbound radioactivity and lysed in 0.2 M NaOH for 15 min. Then the lysate was transferred to a test tube and counted in a gamma counter. Binding activities were also evaluated in the absence or presence of 1 mM unlabeled GA5 or angiopoietin-2. In order to calculate the GA5 binding constant, 1�104 SMMC7721-ExTie2 cells were inoculated in each well and cultured overnight. The next day, cells were washed three times with PBS and blocked with 200 ml blocking buffer (PBS containing 10 mg/ml BSA). After washing three times with PBST, cells were incubated with varying concentrations of serially diluted 125I-GA5 (0-2000 ng/ml) for 30 min at 37 �C and were washed three times with ice-cold PBST to remove unbound radioactivity. The cultured cells were digested with 100 ml of 0.25% trypsin and transferred to a test tube for radioactivity counting.
In vivo biodistribution assay
Four-week-old female athymic mice (BALB/c) were maintained in the Shanghai Cancer Institute Isolation Facility (Shanghai Jiao-Tong University Medical School, Shanghai, China). An athymic mice model bearing SPC-A1 xenograft was established [30]. Either 1 mCi 125I-GA5 or 125I was injected into the lateral tail vein in a total volume of 100 ml PBS. Mice were killed by cervical dislocation at 0.5 h or 4 h after injection and dissected. Tumor tissues and other organs were removed and blotted dry on tissue paper. The wet weight of all samples was recorded, and the radioactivity in each sample was measured with an automated gamma counter. The percentage of injected dose per gram (%ID/g) was calculated according to the injection dose standard curve.
Internalization assay
Fluorescein-isothiocyanate (FITC; Pierce, Rockford, USA) was conjugated to the NH2 terminus of GA5 according to previous report [7]. FITC-labeled peptide was purified by gel filtration with Sephadex G-25 (GE Healthcare Bio-Sciences Corp, Piscataway, USA). The SPC-A1 cells and SMMC7721 cells were cultured on cover slides, then incubated with FITC-labeled GA5 at 37 �C for 10 min and washed three times with PBS. The cells were visualized under a Zeiss Axioskop 2 fluorescence microscope (Zeiss, Oberkochen, Germany).
Preparation of GA5-PEI/DNA
complexes
PEI of 22 kDa (Exgen 500; Fermentas, Hanover, USA) was conjugated with GA5 at the molar ratio of 1:1. The conjugation procedure of GA5-PEI was largely the same as that described previously [32]. Briefly, dithiobis(succinimidylpropionate) (Sigma-Aldrich) was conjugated first with PEI, then with GA5 peptide. The reaction mixture was incubated for 2 h at room temperature, then reaction by-products and DMSO were removed by dialysing. GA5-PEI and plasmid DNA were sterilized by filtration through 0.22 mm filters (Millipore, Billerica, USA). Plasmid DNA was dissolved in small aliquot of distilled water. The PEI cation to DNA anion ratio is presented as the molar ratio of PEI nitrogen to DNA phosphate. The DNA/GA5-PEI complex was prepared by mixing DNA with GA5-PEI for 20 min. The resulting polyplexes were subjected to electrophoresis in 1% agarose containing 0.5 mg/ml ethidium bromide, and approximately 0.5 mg plasmid DNA was loaded into each well.
Targeted reporter gene
delivery in vitro and in vivo
In the in vitro gene delivery assay, 2�104 SMMC7721 and SPC-A1 cells were seeded into 0.5 ml medium in each well of a 24-well plate (Falcon, St. Louis, USA). After cells reached a confluence of approximately 50%, the medium was removed and washed with 0.5 ml PBS, then replaced with 0.5 ml serum-free media containing GA5-PEI/pBK-CMV (pCMV)-luciferase polyplexes or PEI/DNA with the quantity equivalent to 1 mg DNA. Cells were cultured at 37 �C for 4 h. The incubation media were removed and cells were rinsed with 0.5 ml PBS, followed by the addition of 0.5 ml fresh media containing antibiotics and 10% fetal bovine serum. The cells were incubated for another 24 h, and the activity of luciferase was measured in terms of relative light units per milligram protein (RLU/mg). In the in vivo assay, GA5-PEI/pCMV-luciferase polyplexes in a volume of 200 ml were intratumorally injected into athymic mice with SPC-A1 tumor xenograft at a dose equivalent to 50 mg DNA per mouse. The mice were killed 24 h after injection and tumor xenografts, heart, liver, spleen, lung, kidney, and brain were removed and washed three times with 0.1 M PBS (pH 7.4). The expression of luciferase was determined in the tumor as well as in other tissues according to methods reported previously [7]. All experiments were carried out in experimental groups containing at least six mice bearing 500 mm3 tumor. PEI/pCMV-luciferase complexes were used as controls.
Tumor growth inhibition
experiments
Human lung cancer SPC-A1 cells (5�106) were inoculated subcutaneously into 4-week-old female athymic mice (BALB/c). When the tumor size reached 500 mm3, mice were randomly divided into the following five groups with six mice in each group: GA5; PEI/wt p53; GA5-PEI/wt p53; wt p53; and NS. Mice were intratumorally injected with GA5-PEI/wt p53 complexes and other agents every 2 d for a total of eight times. Tumor growth was monitored by measuring the tumor dimensions with a Vernier caliper three times weekly until necrobiosis appeared in tumor xenograft. Tumor volume was calculated according to the formula (V=pab2/6).
Statistical analysis
One-way ANOVA followed by the two-tailed Student's t-test was used for statistical evaluation of differences.
Results
Enzyme-linked immunosorbent
assay and phage recovery assay
After five rounds of screening, 20 phage clones were picked out randomly and amplified for DNA sequencing. Of these, only 17 clones had efficiently inserted peptides, and approximately 35% (6/17) of recovered clones expressed the consensus amino acid sequence NSLSNASEFRAP. Another enriched phage clone (No. 3) displayed a peptide XXGTHGHCQLSH. To assess the specificity of the selected phage, the enriched phage clones No. 46, bearing NSLSNASEFRAP, and No. 3 were amplified for further characterization. Fig. 1(A) illustrates the binding affinity of No. 46 phage clones on various targets by enzyme-linked immunosorbent assay. In phage recovery assay, the binding activity of No. 46 phage clone could be inhibited by Ang-2. However, the insertless phage clone and clone No. 3 did not have such binding specificity [Fig. 1(B-D)]. Based on these data, the peptide clone No. 46 was chosen as our candidate ligand.
Specific binding assay of 125I-labeled GA5 in vitro
and in vivo
In order to label with 125I, tyrosine (Y) was added to the C-terminal of the peptide that was displayed by phage clone No. 46 and was designated GA5 (NSLSNASEFRAPY). As shown in Fig. 2(A), the bound 125I-GA5 radioactivities appeared mainly in SMMC7721-ExTie2 cells, but not in the parent Tie2-negative SMMC7721 cells. In the absence of competitors, approximately 60% of 125I-GA5 was found to localize in the SMMC7721-ExTie2 cells. In the presence of 100-fold molar excess of either Ang-2 or GA5, the bound radioactivity was reduced dramatically to the background level. The dose-response curves of total, specific, and non-specific binding of 125I-GA5 to Tie2 are shown in Fig. 2(B). The specific binding of 125I-GA5 reached a plateau, indicating that it was saturated. The binding constant of radiolabeled GA5 was calculated using Scatchard analysis. The Scatchard plot of GA5 binding to SMMC7721-ExTie2 cells is shown in Fig. 2(C). The Kd value was determined to be (2.10�0.15)�10-8 M. The number of binding sites for the labeled peptide (receptor density) was estimated as (4.52�0.15)�105 per SMMC7721-ExTie2 cell. The biodistribution assay showed that the radioiodine activity level in kidney peaked at 30 min after injection, but declined to one-eighth of its peak by 4 h. However, the tumor uptake of 125I-GA5 was in a different pattern, as shown in Fig. 2(D). The uptake of 125I-GA5 in SPC-A1 xenografts at 0.5 h after injection was up to (8.31�0.46)%ID/g, and declined to (2.41�0.13)%ID/g 4 h later. With the exception of brain, the ratios of radioactivity of tumors to different normal tissues were approximately 3:1. Blocking studies revealed that 100 mg non-radioactive GA5 co-injected with the radiolabeled peptide solution significantly reduced the tumor uptake of radioiodinated GA5 (p<0.05). These data showed that the radioactivity (%ID/g) of tumors was significantly higher than that of other tissues at 4 h (p<0.05) or 0.5 h (p<0.05) after injection except in kidney and blood.
Internalization experiments
To examine whether GA5 can be internalized into Tie2-expressing cells, SPC-A1 and SMMC-7721 cells were incubated with FITC-labeled GA5 peptides, and it was found that the peptides were taken up efficiently by SPC-A1 cells but not SMMC-7721 cells (Fig. 3).
In vitro and in vivo gene
delivery assay
To better understand the effect of GA5 peptide on targeted tumor cells, GA5/PEI conjugate was prepared and pCMV-luciferase was used as a reporter to test its capability for gene delivery. As shown in Fig. 4(A), GA5-PEI/pCMV-luciferase and PEI/pCMV-luciferase could efficiently mediate gene delivery into SPC-A1 and SMMC7721 cells. There was no significant difference in gene delivery in SPC-A1 cells by either GA5-conjugated PEI or PEI alone. However, the luciferase activity in SPC-A1 cells treated with GA5-PEI/pCMV-luciferase was significantly higher than that in Tie2-negative SMMC7721 cells (p<0.05). By in vivo gene delivery assay, the luciferase activity was higher in SPC-A1 tumor xenografts transfected with GA5-PEI/DNA complex than that with PEI/DNA (P<0.05) [Fig. 4(B)]. These data indicated that GA5-PEI vector could mediate gene-targeted delivery.
Targeted gene therapy assay
In vivo gene therapy experiments showed that no difference was observed in tumor growth between untreated mice and control mice treated with irrelevant PEI/DNA complex. Compared with the NS group, the inhibition rate of the GA5-PEI/p53 group was 62.54%. As shown in Fig. 5, intratumoral injection with GA5-PEI/wt p53 complexes could inhibit tumor xenograft growth.
Discussion
Receptor-mediated non-viral gene delivery vectors have been developed to target therapeutic genes into tumor cells through surface receptor. Because of its over-expression in both endothelial cells and certain tumor cells, Tie2 has emerged as a promising target for cancer therapy [21]. To identify a small peptide ligand of Tie2 for targeted gene therapy, we screened a phage display peptide library by competitive biopanning. After five rounds of biopanning, we identified a small peptide, NSLSNASEFRAP, then tyrosine (Y) was added for the purpose of iodination; the peptide NSLSNASEFRAPY was designated GA5. GA5 showed specific binding capability to Tie2 and could be internalized into Tie2-expressing cells. Our previous study revealed that SPC-A1 cells express Tie2 [30]. Therefore, the biodistribution assay was carried out in nude mice bearing human lung adenocarcinoma cell line SPC-A1 xenograft to examine the peptide's targeting ability. After vein injection, we observed higher accumulation of radiolabeled GA5 in tumors than in other organs except kidney. GA5 showed greater specificity for tumor xenografts and higher ratios of tumors to normal tissues at equivalent time points after injection (Fig. 4). Our data showed that GA5 might be an efficient ligand of Tie2 and a novel agent for tumor targeting.
One of the most promising non-viral vectors that has been developed is the polycation PEI. Goula et al used linear low molecular weight PEI (22 kDa)/DNA complexes for systemic application and showed efficient gene delivery, with high gene expression in lung and lower expression in a variety of organs including heart, liver, spleen, and kidney [33]. Li et al reported that GE11, a small peptide ligand of EGFR, conjugated PEI vector can efficiently transfer genes into EGFR over-expressing cells and tumor xenografts [7]. Our results showed that high luciferase activity was observed in SPC-A1 cells expressing Tie2 when transfected with reporter gene pCMV-luciferase by GA5-PEI, but lower luciferase activity was detected in SMMC-7721 cells. In the in vivo gene delivery assay, it was shown that luciferase activity was higher in SPC-A1 tumor xenografts transfected with GA5-PEI/DNA complex than that with PEI/DNA (P<0.05) [Fig. 4(B)]. These results indicated that GA5-PEI could mediate specific delivery of DNA complexes into tumor cells.
In this study, the interesting phenomenon is that the novel vector, GA5-PEI, could successfully deliver reporter gene into SPC-A1 cells but less efficiently into SMMC7721 cells in vitro. Both PEI and GA5-PEI can efficiently mediate reporter gene delivery into SPC-A1 cells, however, in vivo gene delivery assay showed that GA5-PEI, not PEI, could transfer reporter gene into SPC-A1 xenografts. This is most likely due to the generally rapid diffusion and clearance of PEI/DNA polyplexes from circulation and most normal organs that barely express Tie2. In addition, PEI-conjugated GA5 contributed to its stabilization and was specifically accumulated in Tie2-positive cells. In the in vivo gene therapy assay, the inhibition rate of the GA5-PEI/p53 group was 62.54% compared with the normal saline group. GA5-PEI/p53 complexes transferring inhibited tumor xenograft growth and GA5 could be an ideal agent targeting cells that express tie2 receptor.
Acknowledgements
We thank Mrs. Yuyan Zhang for helpful advice in fluorescence microscopy and excellent technical assistance, Dr. Dafang Wan for helpful suggestions, Dr. Wenxin Qin for offering pCDNA3.0 plasmid, Dr. Rong Wang (medical school of Oregon state University, Corvallis, USA) for giving correction highlights.
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