Research Article Received: 27 August 2013
Revised: 18 November 2013
Accepted: 18 November 2013
Published online in Wiley Online Library: 30 January 2014
(wileyonlinelibrary.com) DOI 10.1002/psc.2599
Screening and identification of a specific peptide binding to hepatocellular carcinoma cells from a phage display peptide library Yonge Guo,a Caixia Ma,a Chunyan Li,a Jinling Wu,a Dan Zhang,b Juanjuan Han,a Qixuan Wang,a Jinhui Xu,a Shaoying Lub and Yingchun Houa* To screen and identify the novel probe markers binding hepatocellular carcinoma specifically and sensitively, a phagedisplayed 12-mer peptide library was used to make biopanning with the modified protocols on HepG2 cells. After four rounds of panning, the consensus sequences were obtained, and the PC28, a phage clone with most specific and sensitive binding to HepG2 cells, was identified as the best positive clone. The peptide probe HCSP4 (sequence SLDSTHTHAPWP) was synthesized based on the sequencing result of PC28. The specificity and sensitivity of HCSP4 were primarily analyzed using immunofluorescence, flow cytometry, and other methods. The results show that HCSP4 can bind to hepatocellular carcinoma cells with satisfactory specificity and sensitivity. It may be a promising lead candidate for molecular imaging and targeted drug delivery in the diagnosis and therapy of hepatocellular carcinoma. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. Keywords: hepatocellular carcinoma; phage display; molecular probe; peptide library
196
Introduction
Material and Methods
Hepatocellular carcinoma (HCC) is one of the most common solid malignant tumors worldwide and is often associated with a poor prognosis [1]. The disease has become a significant health burden to human because of the lack of effective treatment. One promising approach to improve treatment efficiency is the targeted therapy using anti-cancer drugs that act on tumor-specific targets. Phage display is a powerful technology that allows the presentation of a large number of proteins or peptides on the surface of filamentous phage [2,3]. The technology provides a means to generate unique peptides that bind any given target [4–8]. Peptides can selectively bind to targets in tumor tissue and can be isolated. Phage display has successfully selected peptides that are specific for tumor cells, such as breast tumor cells [9], bladder tumor cells [10], colon tumor cells [11,12], prostate tumor cells [13], and renal tumor cells [14]. Currently, molecular imaging is commonly used to identify and characterize tumors and other lesions based on their protein expression pattern rather than their macroscopic morphology [3]. Peptides are thought to have advantages over high molecular weight antibodies as imaging probes because they have smaller sizes and better tumor penetration [15,16]. In this regard, peptide probes that can specifically bind to targets would be useful for molecular imaging and early detection of cancer. In the present study, we identified a novel peptide capable of specifically binding to HepG2 cells using the modified subtractive phage display technology. One of our selected peptides, HCSP4, is effective in targeting to HepG2 cells, indicating its potential for use in the early diagnosis and targeted therapy of HCC.
Cell Lines and Cell Culture
J. Pept. Sci. 2014; 20: 196–202
The human HCC cell line HepG2 and the human embryonic kidney cell line HEK293 were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, BRL, Gaithersburg, MD, USA), supplemented with 10% (v/v) fetal bovine serum (Gibco) at 37 °C in a humidified atmosphere of 5% CO2.
Biopanning of a Phage-displayed Peptide Library The panning protocol was performed, as described in the Ph.D.-12 Phage Display Peptide Library Kit manual (New England Biolabs,
* Correspondence to: Yingchun Hou, Department of Cell Biology, College of Life Sciences, Shaanxi Normal University, 199 South Chang’an Road, Xi’an 710062, China. E-mail: [email protected] a Department of Cell Biology, College of Life Sciences, Shaanxi Normal University, 199 South Chang’an Road, Xi’an 710062, China b Department of General Surgery, The First Affiliated Hospital, Xi’an Jiaotong University, 277 West Yanta Road, Xi’an 710061, China Abbreviations: HCC, hepatocellular carcinoma; BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’ medium; pfu, plaque forming unit; ELISA, enzyme-linked immunosorbent assay; IRPs, irrelevant phages; HRP, horseradish peroxidase; HPLC, high-performance liquid chromatography; DAPI, 4′,6diamidino-2-phenylindole; FITC, fluorescein isothiocyanate.
Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
SCREENING AND IDENTIFICATION OF AN HCC PEPTIDE Beverly, MA, USA). HepG2 cells and HEK293 cells were separately seeded into six-well plate (2 × 106 cells/well) and allowed to grow overnight. Cells were blocked at 37 °C for 30 min with DMEM medium containing 1.5% (w/v) bovine serum albumin (BSA). The phage library at 3 × 1011 plaque forming unit (pfu) was incubated with HEK293 cells at 4 °C for 1.5 h. After incubation, culture medium containing the unbound phages was transferred to HepG2 cell monolayer cultures and incubated at 37 °C for 1.5 h. After four rounds of biopanning, the cell-bound phages were amplified and titrated in Escherichia coli ER2738 culture (New England Biolabs). The single-phage plaques were subsequently analyzed. Identification of Positive Phage Clones by Cell Enzyme-linked Immunosorbent Assay (ELISA) HepG2 cells and HEK293 cells were seeded into 96-well plates (2 × 105 cells/well) overnight. Cells were then fixed on 96-well plates by 4% paraformaldehyde for 25 min at room temperature until cells were attached to the plates, and then washed three times with phosphate buffered saline (PBS). Cells were blocked with the blocking buffer (Tris buffered saline with 1.5% w/v BSA) for 1 h. The selected phage clones (1010 pfu/ml) were added to individual wells and incubated at 37 °C for 1 h. PBS and irrelevant phages (IRPs) were used as negative controls. Cells were then washed three times with PBS and incubated with the goat anti-M13 polyclonal antibody (diluted at 1 : 2000 in 1.5% BSA, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 37 °C for 1 h. Subsequently, the horseradish peroxidase (HRP)-labeled rabbit anti-goat IgG antibody (diluted at 1 : 8000 in blocking buffer, Beijing Biosynthesis Biotechnology Co., Ltd., China) was incubated with cells. Finally, the cells were washed with PBS and incubated with the 3,3′,5,5′-tetramethylbenzidine enzyme substrate (Sigma, Sigma-Aldrich. St. Louis, MO 63103, USA) at 37 °C for 15 min. Reaction was terminated by adding 2 M H2SO4 and measured with a microplate reader at 450 nm wave length. Immunofluorescence Assay HepG2 cells and HEK293 cells (2 × 105 cells/well) were cultured on coverslips and fixed with 4% paraformaldehyde at 37 °C for 25 min. Cells were then blocked with PBS containing 1.5% (w/v) BSA for 30 min. The selected positive phage clones (1010 pfu/ml) were incubated with cells at 37 °C for 2.5 h. PBS and IRPs were used as negative controls. After being washed with PBS, the coverslips were incubated with goat anti-M13 polyclonal antibody (working dilution of 1 : 100) at 4 °C overnight. The coverslips were rinsed for three times with PBS and incubated with the fluorescein isothiocyanate (FITC)-labeled rabbit anti-goat IgG antibody (working dilution of 1 : 100, Beijing Biosynthesis Biotechnology Co., Ltd.) at 37 °C for 1 h. Subsequently, the coverslips were rinsed for three times with PBS and stained with 4′,6diamidino-2-phenylindole (DAPI) for nuclear staining. Finally, the coverslips were washed three times with PBS. Images were captured using laser scanning confocal microscope (LSCM, Leica, Germany). Peptide Synthesis
Competitive Inhibition Assay HepG2 cells were seeded into 96-well plates overnight and analyzed as described previously. The candidate peptide HCSP4 and control peptide were then added to each well at concentrations of 0, 100, 200, 300, 400, and 500 μM, incubated with the HepG2 cells at 37 °C for 1.5 h. After being washed for three times with PBS, the cells were incubated with the candidate PC28 phage (1010 pfu/ml) at 37 °C for 2 h. Subsequently, cells were treated as described previously (see section on Identification of Positive Phage Clones by ELISA). The rate of inhibition was calculated using the following formula: the inhibition ratio = (ODcontrol ODphage) / ODcontrol, where ODcontrol and ODphage represent the OD values from no peptide addition groups and phage-binding groups. Flow Cytometry Analysis of Peptide Binding HepG2 cells were plated in 12-well plates and, 24 h later, once the cells reached 90% confluence, incubated with culture medium containing 1.5% BSA at 37 °C for 30 min for blocking and then with FITC-conjugated candidate peptide (0, 2.5, and 5 μM) at 37 °C for 1 h. Control peptide (5 μM) was used as negative control. After being washed three times with PBS, cells were subjected to flow cytometry (Guava easyCyte 8HT, EMD Millipore, USA). Data acquisition and analysis were carried out using the software from FACSCalibur cytometer (Becton Dickinson, USA). The percentage numbers represent the ratio of cells bound by the peptide probe. Fluorescence Imaging of Peptide Binding To further evaluate the affinity of peptide probe HCSP4 binding to HepG2 cells. HepG2 cells and HEK293 cells were all plated on coverslips overnight. Cells were fixed by 4% paraformaldehyde at 37 °C for 15 min. After blocking with 1% (w/v) BSA, cells were then incubated with 5 μM of the FITC-conjugated candidate peptide; control peptide (5 μM) was used as negative control. After being washed three times with PBS, slides were stained with DAPI for nuclear staining and observed using LSCM (Leica). Localization Analysis of the Peptide Probe To determine whether the peptide probe HCSP4 can specifically bind to the cell membrane of HepG2, cells were stained with the Dil membrane probe (Beyotime, Haimen, China) for 15 min at 37 °C, washed three times with PBS, and incubated with FITC-conjugated candidate peptide for 15 min. After washing, the cells were visualized by LSCM (Leica).
Results In Vitro Biopanning Phages specifically binding to HepG2 cells were identified through four rounds of in vitro panning. Table 1 shows an obvious enrichment of phages that are specifically bound to HepG2 cells. After four rounds of screening, the number of phages recovered from HepG2 cells was 53-fold higher (from 1.9 × 107 to 1.0 × 109) than
J. Pept. Sci. 2014; 20: 196–202 Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci
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The candidate peptide translated from the selected positive phage DNA sequence and the control peptide displayed on an irrelevant
phage were synthesized and purified by ChinaPeptides Co., Ltd, Shanghai, China. FITC was conjugated to the N-terminus of each peptide. The sequence and structure of each peptide were characterized by mass spectrometry, and the purity of the peptides was determined by high-performance liquid chromatography (HPLC).
GUO ET AL. Table 1. Input and output phages in each round biopanning Round
Input phages (pfu/ml)a
Output phages (pfu/ml)b
Recovery rate (output/input)c
Table 2. Alignment of phage-displayed peptide sequences selected against HepG2 cells Phage
Peptide number
Peptide sequence
a
PC6,PC26 PC11,PC24 PC16 PC18,PC27,PC28 PC IRPsa
HCSP1 HCSP2 HCSP3 HCSP4 CCSP4
TLADTHTHRPWT LALDTPSHRPWT FWGVTPHGELRS SLDSTHTHAPWP HEDTTGTASYRD
b
a
1 2 3 4
11
1.5 × 10 11 1.5 × 10 11 1.5 × 10 11 1.5 × 10
7
1.9 × 10 8 1.4 × 10 8 1.8 × 10 9 1.0 × 10
- 4
1.3 × 10 - 4 9.3 × 10 - 3 1.2 × 10 - 3 6.7 × 10
Number of phages put into the selection process. Number of phages in the eluate. c Recovery rate of phage clone = output phage/input phage.
that of the first round. The results showed that the output of affinity phages was significantly increased, suggesting an obvious enrichment of phages specifically binding to HepG2 cells. Confirmation of In Vitro Binding by Cellular ELISA After four rounds of panning, 30 blue plaques were randomly selected and individually amplified. Cellular ELISA was performed to study cellular affinity. Of the 30 phage clones, eight phage clones showed higher binding to HepG2 cells when compared with the IRP phage clones and PBS control groups. Furthermore, the PC28 clone appeared to bind most effectively to HepG2 cells than the other clones (Figure 1). Subsequently, the eight positive phage clones were sequenced. Homologous analysis was then performed, and phages with the same sequence were classified. According to the results of DNA sequencing, four different consensus sequences were obtained, and PC28 was the most frequent clone (Table 2). The peptide sequence of HCSP4 appeared three times in the eight positive phage clones. Additionally, complete homology for the entire sequence was not found. Therefore, the phage clone PC28 and its displaying peptide were further investigated. Validation of Phage PC28 by Immunofluorescence Assay Immunofluorescence assay further evaluated the affinity of the PC28 phage clone binding to HepG2 cells. Green
Phage clone displaying the IRPs.
fluorescence was predominantly observed on the cell surfaces of HepG2 cells incubated with PC28 phage clone, whereas only background staining occurred with the control HEK293 cells (Figure 2). To further verify the specificity of the PC28 phage clone, IRP phage clones and PBS treatments were used as negative controls. Similarly, HepG2 cells incubated with IRP phage clones or PBS exhibited weak background staining. The results suggest that the PC28 phage clone could bind specifically to HepG2 cells.
Peptide Synthesis Peptide probe HCSP4 (SLDSTHTHAPWP, based on the sequencing result of PC28 phage) and the irrelevant control peptide probe CCSP4 (HEDTTGTASYRD) were synthesized and conjugated with FITC. The peptide probes were purified to a minimum purity of 95% by HPLC.
Competitive Inhibition Assay To discover whether the synthetic peptide HCSP4 and the selected phage clone competed for the same binding site, competitive inhibition assay was performed. The results showed that the binding of the PC28 phage clone to HepG2 cells was inhibited in a concentration-dependent manner (Figure 3). When the concentrations of peptide HCSP4 increased above 450 μM, the inhibition rate came to a flat phase. However, no significant difference was observed for the control peptide. These data strongly imply that HCSP4 can compete with the binding of the PC28 phage clone to HepG2 cells, which indicated that the binding of phage PC28 to target cells was mediated by the peptide HCSP4.
Flow Cytometry Analysis of Peptide Binding
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Figure 1. Evaluation of the affinity of eight phage clones to HepG2 cells using ELISA. HepG2 cells were seeded into 96-well plates. Phage clones binding to HepG2 cells were treated with the goat anti-M13 and HRP-conjugated rabbit anti-goat IgG antibodies. Plates were read at 450 nm. Phage clones displaying IRPs and PBS treatments were used as negative controls. Triplicate determinations were performed at each data point.
The binding specificity of the FITC-conjugated HCSP4 to HepG2 cells was further validated by flow cytometry analysis. As shown in Figure 4, HepG2 cells were incubated with HCSP4 peptide (0, 2.5, and 5 μM) and control peptide (5 μM), and the numbers represent the percentage of cells bound by peptide. The control peptide showed very little specificity and stained 6.47% of the tumor cells, whereas the HCSP4 peptide stained 17.97% (Figure 4). Therefore, the binding specificity increased gradually by increasing concentrations of peptide HCSP4. However, the control peptide showed weak binding activity to HepG2 cells. These results suggest that the FITC-conjugated candidate peptide could specifically bind to HepG2 cells.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2014; 20: 196–202
SCREENING AND IDENTIFICATION OF AN HCC PEPTIDE
Figure 2. Binding evaluation of the phage PC28 to HepG2 cells was measured by immunofluorescence assay under LSCM. Phage PC28 binds to HepG2 cells (green) but not to control HEK293 cells. Phage displaying irrelevant peptides (IRPs) did not bind to HepG2 cells and HEK293 cells. For negative control groups, HepG2 cells and HEK293 cells were incubated with PBS. Cell nuclei were stained with DAPI (blue). Scale bar 25 μm.
Confirmation Peptide Binding to HepG2 Cells by Fluorescence Imaging
To further characterize whether HCSP4 can specifically bind to the cell surface of HepG2, cell membranes were stained with Dil (Beyotime). As shown in (Figure 6), red fluorescence was predominantly observed on cell membrane of HepG2 cells, and green fluorescence of the FITC-conjugated HCSP4 was indeed localized on cell membrane. Localization analysis was consistent with the results of the fluorescence imaging, which further confirmed that HCSP4 binds to the surfaces of HepG2 cells specifically.
J. Pept. Sci. 2014; 20: 196–202 Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci
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Fluorescence imaging further confirmed the binding ability and specificity of the synthetic HCSP4 peptide. Green fluorescence was dominantly observed on the HepG2 cells [Figure 5(A)]. In contrast, only background staining occurred in the HEK293 cells, which might have resulted from a lack of HCSP4 receptors. Furthermore, the control peptide CCSP4 showed minimal binding to the HepG2 [Figure 5(C)] and HEK293 cells [Figure 5(D)]. Together, these results strongly demonstrate that HCSP4 can bind to HepG2 cells with satisfied specificity and sensitivity.
Localization Analysis of the Peptide Probe
GUO ET AL.
Figure 3. Competitive inhibition of the phage PC28 binding to HepG2 cells by the synthetic peptide HCSP4. The average inhibition rates at different concentrations of the peptide were shown. When the concentration of peptide HCSP4 was increased above 100 μM, a significant inhibition occurred. The control peptide was used as a negative control. Data are expressed as the mean ± SD of triplicates from three independent experiments.
Discussion Hepatocellular carcinoma, a form of cancer originating in liver cells, has been the third leading cause of cancer-related death [17]. HCC is a challenging malignancy with high patient mortality rates. Although therapies are available, drawbacks such as cytotoxicity have prompted researchers to seek more effective
treatment [16]. Thus, it is urgent to find an innovative method for early detection of HCC. Cancer cells often display high numbers of certain cell surface molecules, such as tumor-associated antigens or specific receptors, that occur in normal tissues and represent potential targets for tumor diagnosis and treatment [3]. Phage display is a powerful combinatorial technique for specific binding to a desired cell type. Peptides with specificity for a particular cell type can be identified after four rounds of screening [18,19]. Several previous reports demonstrated that some specific receptors show high expressions levels on the surface of tumor cells and they can selectively bind to certain ligand [20]. Therefore, the screening and identification of peptides that specifically bind to the HepG2 cells will aid in the development of novel probes for HCC detection and therapy. In this study, phages specifically binding to HepG2 cells were identified through four rounds of panning. However, in our panning protocol, we used BRASIL assay to select some phages specifically binding to HepG2 cells. After four rounds of biopanning, the phage recovery rate gradually increased, and positive phage clones were effectively enriched (Table 1). We randomly selected 30 phage clones for further characterization. Subsequently, the phage clones were further tested using cellular ELISA to confirm their specific binding to HepG2 cells. Of the 30 phage clones, eight phage clones showed higher binding than any of the other clones and were sequenced. A multiple sequence alignment showed that the sequence did not exhibit homology to the sequences of any characterized proteins in various protein databases. This finding demonstrates that HCSP4 is a novel peptide that specifically binds to HepG2 cells (Figure 1). Immunofluorescence assay further evaluated the affinity of PC28
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Figure 4. Binding specificity of peptide HCSP4 to HepG2 cells was measured by flow cytometry. (A) HepG2 cells were treated with PBS. (B) HepG2 cells were incubated with 2.5 μM peptide HCSP4. (C) HepG2 cells were incubated with 5 μM peptide HCSP4. (D) HepG2 cells were incubated with 5 μM control peptide. The percentage numbers represent the ratio of cells bound by the peptide probe.
wileyonlinelibrary.com/journal/jpepsci Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2014; 20: 196–202
SCREENING AND IDENTIFICATION OF AN HCC PEPTIDE
Figure 5. Fluorescence imaging of peptide probe HCSP4 binding to HepG2 cells under LSCM. The FITC-labeled synthetic peptide HCSP4 binds to HepG2 cells (A) but not to control (B) HEK293 cells. At the same time, the FITC-labeled control peptide CCSP4 shows weak binding to (C) HepG2 cells and (D) HEK293 cells. Cell nuclei were stained blue with DAPI. Scale bar 25 μm.
Figure 6. Localization analysis of peptide probe HCSP4 binding to HepG2 cells under LSCM. (A) The FITC-labeled HCSP4 peptide was incubated with HepG2 cells (green). (B) The cell membrane was stained with Dil (red). (C) The merged photos show that HCSP4 binds to the surfaces of HepG2 cells specifically. Scale bar 7.5 μm.
cytometry analysis further confirmed that HCSP4 can specifically bind to HepG2 cells based on high levels fluorescence (Figure 4). To further verify the specificity of the HCSP4 peptide, HepG2 and HEK293 cells were incubated with FITC-conjugated peptides; the results indicated that green fluorescence was dominantly observed on cell surface of HepG2 cells [Figure 5(A)]. However, little
J. Pept. Sci. 2014; 20: 196–202 Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci
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phage clone binding to HepG2 cells and not the control HEK293 cells (Figure 2). To discover whether the synthetic peptide HCSP4 and the selected phage clone competed for the same binding site, competitive inhibition assay was performed. These results confirmed that the binding of phage PC28 to target cells was mediated by the peptide HCSP4 (Figure 3). The results of flow
GUO ET AL. binding was observed in the HEK293 cells, which might have resulted from a lack of HCSP4 receptors. We guessed that the binding specificity mechanism could involve several membrane receptors. To investigate the detailed distribution of the HCSP4 binding receptors on HepG2 cells, cell membranes were stained with Dil (Beyotime). The results showed that the HCSP4 binding receptors were really localized on the cell membrane of HepG2 cells (Figure 6). However, further studies are needed to validate the specific binding receptors. Together, these results provide strong evidence that HCSP4 binds specifically to the HepG2 cells and may be a useful targeting probe for the diagnosis and treatment of HCC. Here, we report the selection of a peptide specific for HepG2 cells using a phage display peptide library. Our results indicate that the HCSP4 peptide could specifically bind to HepG2 cells with high affinity. Experiments evaluating the characteristics of the selected peptide conjugated with FITC as an imaging probe are currently underway, and the results of which indicate that HCSP4 may act as a useful diagnostic molecular imaging probe for detecting HCC. Further studies are needed to investigate the binding specificity of HCSP4 to human hepatocarcinoma tissues and to determine whether HCSP4 can inhibit cancer migration and growth in animal tumor models. Thus, the peptide HCSP4 appeared to possess potential applications in the diagnosis and therapy of HCC. Acknowledgement This work was supported by National Natural Science Foundation of China (no. 81172359).
References 1 Yang X, Xu Y, Guo F, Ning Y, Zhi X, Yin L, Li X. Hydroxysteroid sulfotransferase SULT2B1b promotes hepatocellular carcinoma cells proliferation in vitro and in vivo. PLoS One 2013; 8: e60853. 2 Scott JK, Smith GP. Searching for peptide ligands with an epitope library. Science 1990; 249: 386–390. 3 Tang B, Li Z, Huang D, Zheng L, Li Q. Screening of a specific peptide binding to VPAC1 receptor from a phage display peptide library. PLoS One 2013; 8: e54264. 4 Chen K, Yap LP, Park R, Hui X, Wu K, Fan D, Chen X, Conti PS. A Cy5.5labeled phage-displayed peptide probe for near-infrared fluorescence imaging of tumor vasculature in living mice. Amino Acids 2012; 42: 1329–1337.
5 Estephan E, Larroque C, Bec N, Martineau P, Cuisinier FJG, Cloitre T, Gergely C. Selection and mass spectrometry characterization of peptides targeting semiconductor surfaces. Biotechnol. Bioeng. 2009; 104: 1121–1131. 6 Nicol CG, Denby L, Lopez-Franco O, Masson R, Halliday CA, Nicklin SA, Kritz A, Work LM, Baker AH. Use of in vivo phage display to engineer novel adenoviruses for targeted delivery to the cardiac vasculature. FEBS Lett. 2009; 583: 2100–2107. 7 Whaley SR, English DS, Hu EL, Barbara PF, Belcher AM. Selection of peptides with SC binding specificity for directed nanocrystal assembly. Nature 2000; 405: 665–668. 8 Maruta F, Parker AL, Fisher KD, Murray PG, Kerr DJ, Seymour LW. Use of a phage display library to identify oligopeptides binding to the lumenal surface of polarized endothelium by ex vivo perfusion of human umbilical veins. J. Drug Target. 2003; 11: 53–59. 9 Siebke C, James TC, Cummins R, O’Grady T, Kay E, Bond U. Phage display biopanning identifies the translation initiation and elongation factors (IF1alpha-3 and eIF-3) as components of Hsp70-peptide complexes in breast tumour cells. Cell Stress Chaperones 2012; 17: 145–156. 10 Zhang H, Aina OH, Lam KS, de Vere White R, Evans C, Henderson P, Lara PN, Wang X, Bassuk JA, Pan CX. Identification of a bladder cancer-specific ligand using a combinatorial chemistry approach. Urol. Oncol. 2012; 30: 635–645. 11 Kelly KA, Jones DA. Isolation of a colon tumor specific binding peptide using phage display selection. Neoplasia 2003; 5: 437–444. 12 Joshi BP, Liu Z, Elahi SF, Appelman HD, Wang TD. Near-infraredlabeled peptide multimer functions as phage mimic for high affinity, specific targeting of colonic adenomas in vivo. Gastrointest. Endosc. 2012; 76: 1197–2061. 13 Wang W, Chen X, Li T, Li Y, Wang R, He D, Luo W, Li X, Wu X. Screening a phage display library for a novel FGF8b-binding peptide with antitumor effect on prostate cancer. Exp. Cell Res. 2013; 319: 1156–1164. 14 Bussolati B, Grange C, Tei L, Deregibus MC, Ercolani M, Aime S, Camussi G. Targeting of human renal tumor-derived endothelial cells with peptides obtained by phage display. J. Mol. Med. 2007; 85: 897–906. 15 He X, Na MH, Kim JS, Lee GY, Park JY, Hoffman AS, Nam JO, Han SE, Sim GY, Oh YK, Kim IS, Lee BH. A novel peptide probe for imaging and targeted delivery of liposomal doxorubicin to lung tumor. Mol. Pharm. 2011; 8: 430–438. 16 Filfil R, Paul-Roc B, Cantin C, Iqbal U, Tolkatchev D, Vinogradova A, Xu P, Ni F, O’Connor-McCourt MD, Lenferink AE. Molecular imaging of breast tumors using a near-infrared fluorescently labeled clusterin binding peptide. Int. J. Cancer 2012; 131: E681–E692. 17 Huang YH, Lin KH, Chen HC, Chang ML, Hsu CW, Lai MW, Chen TC, Lee WC, Tseng YH, Yeh CT. Identification of postoperative prognostic microRNA predictors in hepatocellular carcinoma. PLoS One 2012; 7: e37188. 18 Gray BP, Li S, Brown KC. From phage display to nanoparticle delivery: functionalizing liposomes with multivalent peptides improves targeting to a cancer biomarker. Bioconjug. Chem. 2013; 24: 85–96. 19 Bratkovic T. Progress in phage display: evolution of the technique and its application. Cell. Mol. Life Sci. 2009; 67: 749–767. 20 Thomas M. Molecular targeted therapy for hepatocellular carcinoma. J. Gastroenterol. 2009; 44: 136–141.
202 wileyonlinelibrary.com/journal/jpepsci Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2014; 20: 196–202
Revised: 18 November 2013
Accepted: 18 November 2013
Published online in Wiley Online Library: 30 January 2014
(wileyonlinelibrary.com) DOI 10.1002/psc.2599
Screening and identification of a specific peptide binding to hepatocellular carcinoma cells from a phage display peptide library Yonge Guo,a Caixia Ma,a Chunyan Li,a Jinling Wu,a Dan Zhang,b Juanjuan Han,a Qixuan Wang,a Jinhui Xu,a Shaoying Lub and Yingchun Houa* To screen and identify the novel probe markers binding hepatocellular carcinoma specifically and sensitively, a phagedisplayed 12-mer peptide library was used to make biopanning with the modified protocols on HepG2 cells. After four rounds of panning, the consensus sequences were obtained, and the PC28, a phage clone with most specific and sensitive binding to HepG2 cells, was identified as the best positive clone. The peptide probe HCSP4 (sequence SLDSTHTHAPWP) was synthesized based on the sequencing result of PC28. The specificity and sensitivity of HCSP4 were primarily analyzed using immunofluorescence, flow cytometry, and other methods. The results show that HCSP4 can bind to hepatocellular carcinoma cells with satisfactory specificity and sensitivity. It may be a promising lead candidate for molecular imaging and targeted drug delivery in the diagnosis and therapy of hepatocellular carcinoma. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. Keywords: hepatocellular carcinoma; phage display; molecular probe; peptide library
196
Introduction
Material and Methods
Hepatocellular carcinoma (HCC) is one of the most common solid malignant tumors worldwide and is often associated with a poor prognosis [1]. The disease has become a significant health burden to human because of the lack of effective treatment. One promising approach to improve treatment efficiency is the targeted therapy using anti-cancer drugs that act on tumor-specific targets. Phage display is a powerful technology that allows the presentation of a large number of proteins or peptides on the surface of filamentous phage [2,3]. The technology provides a means to generate unique peptides that bind any given target [4–8]. Peptides can selectively bind to targets in tumor tissue and can be isolated. Phage display has successfully selected peptides that are specific for tumor cells, such as breast tumor cells [9], bladder tumor cells [10], colon tumor cells [11,12], prostate tumor cells [13], and renal tumor cells [14]. Currently, molecular imaging is commonly used to identify and characterize tumors and other lesions based on their protein expression pattern rather than their macroscopic morphology [3]. Peptides are thought to have advantages over high molecular weight antibodies as imaging probes because they have smaller sizes and better tumor penetration [15,16]. In this regard, peptide probes that can specifically bind to targets would be useful for molecular imaging and early detection of cancer. In the present study, we identified a novel peptide capable of specifically binding to HepG2 cells using the modified subtractive phage display technology. One of our selected peptides, HCSP4, is effective in targeting to HepG2 cells, indicating its potential for use in the early diagnosis and targeted therapy of HCC.
Cell Lines and Cell Culture
J. Pept. Sci. 2014; 20: 196–202
The human HCC cell line HepG2 and the human embryonic kidney cell line HEK293 were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, BRL, Gaithersburg, MD, USA), supplemented with 10% (v/v) fetal bovine serum (Gibco) at 37 °C in a humidified atmosphere of 5% CO2.
Biopanning of a Phage-displayed Peptide Library The panning protocol was performed, as described in the Ph.D.-12 Phage Display Peptide Library Kit manual (New England Biolabs,
* Correspondence to: Yingchun Hou, Department of Cell Biology, College of Life Sciences, Shaanxi Normal University, 199 South Chang’an Road, Xi’an 710062, China. E-mail: [email protected] a Department of Cell Biology, College of Life Sciences, Shaanxi Normal University, 199 South Chang’an Road, Xi’an 710062, China b Department of General Surgery, The First Affiliated Hospital, Xi’an Jiaotong University, 277 West Yanta Road, Xi’an 710061, China Abbreviations: HCC, hepatocellular carcinoma; BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’ medium; pfu, plaque forming unit; ELISA, enzyme-linked immunosorbent assay; IRPs, irrelevant phages; HRP, horseradish peroxidase; HPLC, high-performance liquid chromatography; DAPI, 4′,6diamidino-2-phenylindole; FITC, fluorescein isothiocyanate.
Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.
SCREENING AND IDENTIFICATION OF AN HCC PEPTIDE Beverly, MA, USA). HepG2 cells and HEK293 cells were separately seeded into six-well plate (2 × 106 cells/well) and allowed to grow overnight. Cells were blocked at 37 °C for 30 min with DMEM medium containing 1.5% (w/v) bovine serum albumin (BSA). The phage library at 3 × 1011 plaque forming unit (pfu) was incubated with HEK293 cells at 4 °C for 1.5 h. After incubation, culture medium containing the unbound phages was transferred to HepG2 cell monolayer cultures and incubated at 37 °C for 1.5 h. After four rounds of biopanning, the cell-bound phages were amplified and titrated in Escherichia coli ER2738 culture (New England Biolabs). The single-phage plaques were subsequently analyzed. Identification of Positive Phage Clones by Cell Enzyme-linked Immunosorbent Assay (ELISA) HepG2 cells and HEK293 cells were seeded into 96-well plates (2 × 105 cells/well) overnight. Cells were then fixed on 96-well plates by 4% paraformaldehyde for 25 min at room temperature until cells were attached to the plates, and then washed three times with phosphate buffered saline (PBS). Cells were blocked with the blocking buffer (Tris buffered saline with 1.5% w/v BSA) for 1 h. The selected phage clones (1010 pfu/ml) were added to individual wells and incubated at 37 °C for 1 h. PBS and irrelevant phages (IRPs) were used as negative controls. Cells were then washed three times with PBS and incubated with the goat anti-M13 polyclonal antibody (diluted at 1 : 2000 in 1.5% BSA, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 37 °C for 1 h. Subsequently, the horseradish peroxidase (HRP)-labeled rabbit anti-goat IgG antibody (diluted at 1 : 8000 in blocking buffer, Beijing Biosynthesis Biotechnology Co., Ltd., China) was incubated with cells. Finally, the cells were washed with PBS and incubated with the 3,3′,5,5′-tetramethylbenzidine enzyme substrate (Sigma, Sigma-Aldrich. St. Louis, MO 63103, USA) at 37 °C for 15 min. Reaction was terminated by adding 2 M H2SO4 and measured with a microplate reader at 450 nm wave length. Immunofluorescence Assay HepG2 cells and HEK293 cells (2 × 105 cells/well) were cultured on coverslips and fixed with 4% paraformaldehyde at 37 °C for 25 min. Cells were then blocked with PBS containing 1.5% (w/v) BSA for 30 min. The selected positive phage clones (1010 pfu/ml) were incubated with cells at 37 °C for 2.5 h. PBS and IRPs were used as negative controls. After being washed with PBS, the coverslips were incubated with goat anti-M13 polyclonal antibody (working dilution of 1 : 100) at 4 °C overnight. The coverslips were rinsed for three times with PBS and incubated with the fluorescein isothiocyanate (FITC)-labeled rabbit anti-goat IgG antibody (working dilution of 1 : 100, Beijing Biosynthesis Biotechnology Co., Ltd.) at 37 °C for 1 h. Subsequently, the coverslips were rinsed for three times with PBS and stained with 4′,6diamidino-2-phenylindole (DAPI) for nuclear staining. Finally, the coverslips were washed three times with PBS. Images were captured using laser scanning confocal microscope (LSCM, Leica, Germany). Peptide Synthesis
Competitive Inhibition Assay HepG2 cells were seeded into 96-well plates overnight and analyzed as described previously. The candidate peptide HCSP4 and control peptide were then added to each well at concentrations of 0, 100, 200, 300, 400, and 500 μM, incubated with the HepG2 cells at 37 °C for 1.5 h. After being washed for three times with PBS, the cells were incubated with the candidate PC28 phage (1010 pfu/ml) at 37 °C for 2 h. Subsequently, cells were treated as described previously (see section on Identification of Positive Phage Clones by ELISA). The rate of inhibition was calculated using the following formula: the inhibition ratio = (ODcontrol ODphage) / ODcontrol, where ODcontrol and ODphage represent the OD values from no peptide addition groups and phage-binding groups. Flow Cytometry Analysis of Peptide Binding HepG2 cells were plated in 12-well plates and, 24 h later, once the cells reached 90% confluence, incubated with culture medium containing 1.5% BSA at 37 °C for 30 min for blocking and then with FITC-conjugated candidate peptide (0, 2.5, and 5 μM) at 37 °C for 1 h. Control peptide (5 μM) was used as negative control. After being washed three times with PBS, cells were subjected to flow cytometry (Guava easyCyte 8HT, EMD Millipore, USA). Data acquisition and analysis were carried out using the software from FACSCalibur cytometer (Becton Dickinson, USA). The percentage numbers represent the ratio of cells bound by the peptide probe. Fluorescence Imaging of Peptide Binding To further evaluate the affinity of peptide probe HCSP4 binding to HepG2 cells. HepG2 cells and HEK293 cells were all plated on coverslips overnight. Cells were fixed by 4% paraformaldehyde at 37 °C for 15 min. After blocking with 1% (w/v) BSA, cells were then incubated with 5 μM of the FITC-conjugated candidate peptide; control peptide (5 μM) was used as negative control. After being washed three times with PBS, slides were stained with DAPI for nuclear staining and observed using LSCM (Leica). Localization Analysis of the Peptide Probe To determine whether the peptide probe HCSP4 can specifically bind to the cell membrane of HepG2, cells were stained with the Dil membrane probe (Beyotime, Haimen, China) for 15 min at 37 °C, washed three times with PBS, and incubated with FITC-conjugated candidate peptide for 15 min. After washing, the cells were visualized by LSCM (Leica).
Results In Vitro Biopanning Phages specifically binding to HepG2 cells were identified through four rounds of in vitro panning. Table 1 shows an obvious enrichment of phages that are specifically bound to HepG2 cells. After four rounds of screening, the number of phages recovered from HepG2 cells was 53-fold higher (from 1.9 × 107 to 1.0 × 109) than
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The candidate peptide translated from the selected positive phage DNA sequence and the control peptide displayed on an irrelevant
phage were synthesized and purified by ChinaPeptides Co., Ltd, Shanghai, China. FITC was conjugated to the N-terminus of each peptide. The sequence and structure of each peptide were characterized by mass spectrometry, and the purity of the peptides was determined by high-performance liquid chromatography (HPLC).
GUO ET AL. Table 1. Input and output phages in each round biopanning Round
Input phages (pfu/ml)a
Output phages (pfu/ml)b
Recovery rate (output/input)c
Table 2. Alignment of phage-displayed peptide sequences selected against HepG2 cells Phage
Peptide number
Peptide sequence
a
PC6,PC26 PC11,PC24 PC16 PC18,PC27,PC28 PC IRPsa
HCSP1 HCSP2 HCSP3 HCSP4 CCSP4
TLADTHTHRPWT LALDTPSHRPWT FWGVTPHGELRS SLDSTHTHAPWP HEDTTGTASYRD
b
a
1 2 3 4
11
1.5 × 10 11 1.5 × 10 11 1.5 × 10 11 1.5 × 10
7
1.9 × 10 8 1.4 × 10 8 1.8 × 10 9 1.0 × 10
- 4
1.3 × 10 - 4 9.3 × 10 - 3 1.2 × 10 - 3 6.7 × 10
Number of phages put into the selection process. Number of phages in the eluate. c Recovery rate of phage clone = output phage/input phage.
that of the first round. The results showed that the output of affinity phages was significantly increased, suggesting an obvious enrichment of phages specifically binding to HepG2 cells. Confirmation of In Vitro Binding by Cellular ELISA After four rounds of panning, 30 blue plaques were randomly selected and individually amplified. Cellular ELISA was performed to study cellular affinity. Of the 30 phage clones, eight phage clones showed higher binding to HepG2 cells when compared with the IRP phage clones and PBS control groups. Furthermore, the PC28 clone appeared to bind most effectively to HepG2 cells than the other clones (Figure 1). Subsequently, the eight positive phage clones were sequenced. Homologous analysis was then performed, and phages with the same sequence were classified. According to the results of DNA sequencing, four different consensus sequences were obtained, and PC28 was the most frequent clone (Table 2). The peptide sequence of HCSP4 appeared three times in the eight positive phage clones. Additionally, complete homology for the entire sequence was not found. Therefore, the phage clone PC28 and its displaying peptide were further investigated. Validation of Phage PC28 by Immunofluorescence Assay Immunofluorescence assay further evaluated the affinity of the PC28 phage clone binding to HepG2 cells. Green
Phage clone displaying the IRPs.
fluorescence was predominantly observed on the cell surfaces of HepG2 cells incubated with PC28 phage clone, whereas only background staining occurred with the control HEK293 cells (Figure 2). To further verify the specificity of the PC28 phage clone, IRP phage clones and PBS treatments were used as negative controls. Similarly, HepG2 cells incubated with IRP phage clones or PBS exhibited weak background staining. The results suggest that the PC28 phage clone could bind specifically to HepG2 cells.
Peptide Synthesis Peptide probe HCSP4 (SLDSTHTHAPWP, based on the sequencing result of PC28 phage) and the irrelevant control peptide probe CCSP4 (HEDTTGTASYRD) were synthesized and conjugated with FITC. The peptide probes were purified to a minimum purity of 95% by HPLC.
Competitive Inhibition Assay To discover whether the synthetic peptide HCSP4 and the selected phage clone competed for the same binding site, competitive inhibition assay was performed. The results showed that the binding of the PC28 phage clone to HepG2 cells was inhibited in a concentration-dependent manner (Figure 3). When the concentrations of peptide HCSP4 increased above 450 μM, the inhibition rate came to a flat phase. However, no significant difference was observed for the control peptide. These data strongly imply that HCSP4 can compete with the binding of the PC28 phage clone to HepG2 cells, which indicated that the binding of phage PC28 to target cells was mediated by the peptide HCSP4.
Flow Cytometry Analysis of Peptide Binding
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Figure 1. Evaluation of the affinity of eight phage clones to HepG2 cells using ELISA. HepG2 cells were seeded into 96-well plates. Phage clones binding to HepG2 cells were treated with the goat anti-M13 and HRP-conjugated rabbit anti-goat IgG antibodies. Plates were read at 450 nm. Phage clones displaying IRPs and PBS treatments were used as negative controls. Triplicate determinations were performed at each data point.
The binding specificity of the FITC-conjugated HCSP4 to HepG2 cells was further validated by flow cytometry analysis. As shown in Figure 4, HepG2 cells were incubated with HCSP4 peptide (0, 2.5, and 5 μM) and control peptide (5 μM), and the numbers represent the percentage of cells bound by peptide. The control peptide showed very little specificity and stained 6.47% of the tumor cells, whereas the HCSP4 peptide stained 17.97% (Figure 4). Therefore, the binding specificity increased gradually by increasing concentrations of peptide HCSP4. However, the control peptide showed weak binding activity to HepG2 cells. These results suggest that the FITC-conjugated candidate peptide could specifically bind to HepG2 cells.
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SCREENING AND IDENTIFICATION OF AN HCC PEPTIDE
Figure 2. Binding evaluation of the phage PC28 to HepG2 cells was measured by immunofluorescence assay under LSCM. Phage PC28 binds to HepG2 cells (green) but not to control HEK293 cells. Phage displaying irrelevant peptides (IRPs) did not bind to HepG2 cells and HEK293 cells. For negative control groups, HepG2 cells and HEK293 cells were incubated with PBS. Cell nuclei were stained with DAPI (blue). Scale bar 25 μm.
Confirmation Peptide Binding to HepG2 Cells by Fluorescence Imaging
To further characterize whether HCSP4 can specifically bind to the cell surface of HepG2, cell membranes were stained with Dil (Beyotime). As shown in (Figure 6), red fluorescence was predominantly observed on cell membrane of HepG2 cells, and green fluorescence of the FITC-conjugated HCSP4 was indeed localized on cell membrane. Localization analysis was consistent with the results of the fluorescence imaging, which further confirmed that HCSP4 binds to the surfaces of HepG2 cells specifically.
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Fluorescence imaging further confirmed the binding ability and specificity of the synthetic HCSP4 peptide. Green fluorescence was dominantly observed on the HepG2 cells [Figure 5(A)]. In contrast, only background staining occurred in the HEK293 cells, which might have resulted from a lack of HCSP4 receptors. Furthermore, the control peptide CCSP4 showed minimal binding to the HepG2 [Figure 5(C)] and HEK293 cells [Figure 5(D)]. Together, these results strongly demonstrate that HCSP4 can bind to HepG2 cells with satisfied specificity and sensitivity.
Localization Analysis of the Peptide Probe
GUO ET AL.
Figure 3. Competitive inhibition of the phage PC28 binding to HepG2 cells by the synthetic peptide HCSP4. The average inhibition rates at different concentrations of the peptide were shown. When the concentration of peptide HCSP4 was increased above 100 μM, a significant inhibition occurred. The control peptide was used as a negative control. Data are expressed as the mean ± SD of triplicates from three independent experiments.
Discussion Hepatocellular carcinoma, a form of cancer originating in liver cells, has been the third leading cause of cancer-related death [17]. HCC is a challenging malignancy with high patient mortality rates. Although therapies are available, drawbacks such as cytotoxicity have prompted researchers to seek more effective
treatment [16]. Thus, it is urgent to find an innovative method for early detection of HCC. Cancer cells often display high numbers of certain cell surface molecules, such as tumor-associated antigens or specific receptors, that occur in normal tissues and represent potential targets for tumor diagnosis and treatment [3]. Phage display is a powerful combinatorial technique for specific binding to a desired cell type. Peptides with specificity for a particular cell type can be identified after four rounds of screening [18,19]. Several previous reports demonstrated that some specific receptors show high expressions levels on the surface of tumor cells and they can selectively bind to certain ligand [20]. Therefore, the screening and identification of peptides that specifically bind to the HepG2 cells will aid in the development of novel probes for HCC detection and therapy. In this study, phages specifically binding to HepG2 cells were identified through four rounds of panning. However, in our panning protocol, we used BRASIL assay to select some phages specifically binding to HepG2 cells. After four rounds of biopanning, the phage recovery rate gradually increased, and positive phage clones were effectively enriched (Table 1). We randomly selected 30 phage clones for further characterization. Subsequently, the phage clones were further tested using cellular ELISA to confirm their specific binding to HepG2 cells. Of the 30 phage clones, eight phage clones showed higher binding than any of the other clones and were sequenced. A multiple sequence alignment showed that the sequence did not exhibit homology to the sequences of any characterized proteins in various protein databases. This finding demonstrates that HCSP4 is a novel peptide that specifically binds to HepG2 cells (Figure 1). Immunofluorescence assay further evaluated the affinity of PC28
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Figure 4. Binding specificity of peptide HCSP4 to HepG2 cells was measured by flow cytometry. (A) HepG2 cells were treated with PBS. (B) HepG2 cells were incubated with 2.5 μM peptide HCSP4. (C) HepG2 cells were incubated with 5 μM peptide HCSP4. (D) HepG2 cells were incubated with 5 μM control peptide. The percentage numbers represent the ratio of cells bound by the peptide probe.
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SCREENING AND IDENTIFICATION OF AN HCC PEPTIDE
Figure 5. Fluorescence imaging of peptide probe HCSP4 binding to HepG2 cells under LSCM. The FITC-labeled synthetic peptide HCSP4 binds to HepG2 cells (A) but not to control (B) HEK293 cells. At the same time, the FITC-labeled control peptide CCSP4 shows weak binding to (C) HepG2 cells and (D) HEK293 cells. Cell nuclei were stained blue with DAPI. Scale bar 25 μm.
Figure 6. Localization analysis of peptide probe HCSP4 binding to HepG2 cells under LSCM. (A) The FITC-labeled HCSP4 peptide was incubated with HepG2 cells (green). (B) The cell membrane was stained with Dil (red). (C) The merged photos show that HCSP4 binds to the surfaces of HepG2 cells specifically. Scale bar 7.5 μm.
cytometry analysis further confirmed that HCSP4 can specifically bind to HepG2 cells based on high levels fluorescence (Figure 4). To further verify the specificity of the HCSP4 peptide, HepG2 and HEK293 cells were incubated with FITC-conjugated peptides; the results indicated that green fluorescence was dominantly observed on cell surface of HepG2 cells [Figure 5(A)]. However, little
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phage clone binding to HepG2 cells and not the control HEK293 cells (Figure 2). To discover whether the synthetic peptide HCSP4 and the selected phage clone competed for the same binding site, competitive inhibition assay was performed. These results confirmed that the binding of phage PC28 to target cells was mediated by the peptide HCSP4 (Figure 3). The results of flow
GUO ET AL. binding was observed in the HEK293 cells, which might have resulted from a lack of HCSP4 receptors. We guessed that the binding specificity mechanism could involve several membrane receptors. To investigate the detailed distribution of the HCSP4 binding receptors on HepG2 cells, cell membranes were stained with Dil (Beyotime). The results showed that the HCSP4 binding receptors were really localized on the cell membrane of HepG2 cells (Figure 6). However, further studies are needed to validate the specific binding receptors. Together, these results provide strong evidence that HCSP4 binds specifically to the HepG2 cells and may be a useful targeting probe for the diagnosis and treatment of HCC. Here, we report the selection of a peptide specific for HepG2 cells using a phage display peptide library. Our results indicate that the HCSP4 peptide could specifically bind to HepG2 cells with high affinity. Experiments evaluating the characteristics of the selected peptide conjugated with FITC as an imaging probe are currently underway, and the results of which indicate that HCSP4 may act as a useful diagnostic molecular imaging probe for detecting HCC. Further studies are needed to investigate the binding specificity of HCSP4 to human hepatocarcinoma tissues and to determine whether HCSP4 can inhibit cancer migration and growth in animal tumor models. Thus, the peptide HCSP4 appeared to possess potential applications in the diagnosis and therapy of HCC. Acknowledgement This work was supported by National Natural Science Foundation of China (no. 81172359).
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