Cell lines and culture
The human epithelial cell line CACO-2 without mutation of K-ras, human colon cancer cell line SW480 with K-ras mutation , the human non-small cell lung cancer cell line A549, human hepatoma cell line Huh7, human glioma cell line U251, normal human lung epithelial cell line BEAS-2B and the normal colonic cell line CCD841 were purchased from the Chinese Academy of Sciences Cell Bank. The cell lines were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/ml penicillin G, and 100 μg/ml streptomycin under atmospheric conditions of 5% CO2 at 37 °C.
Construction of prokaryotic expression plasmids
The anti-p21Ras scFv was constructed previously in our laboratory . The RGD4C peptide (ACDCRGDCFCG) was developed with phage display technology . The anti-p21Ras scFv gene and RGD4C gene were linked genetically and then inserted into the prokaryotic expression plasmid pET-28a (+) between the BamH I and Hind III sites. The pET-28a (+) expression vector contains two 6 × His tags to allow immobilized metal ion affinity purification. Recombinant plasmids were sequenced for identification (Qingke, China). Four prokaryotic expression plasmids were constructed: p-scFv, p-RGD4C-scFv, p-RGD4C-linker-scFv, and p-RGD4C-EGFP.
Expression and purification of fusion proteins
The recombinant expression plasmids were transformed into Escherichia coli BL21 (DE3) and selected with kanamycin. After PCR identification, a single positive colony was inoculated into 50 mL of LB medium and grown at 37 °C. The fusion protein was expressed inducibly with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 5 h at 22 °C. E. coli BL21 (DE3) was collected by centrifugation at 12,000 rpm for 20 min and ultrasonicated. The supernatant contained soluble protein, and the precipitate contained inclusion body protein. The soluble recombinant protein and inclusion body protein were collected by bacterial sonication in a bacterial lysis buffer (100 mM sodium chloride, 1 mM EDTA, and 50 mM Tris-HCl buffer, pH 8.0), followed by centrifugation (12,000 rpm, 20 min, 4 °C). The insoluble protein fraction was washed 1 time with inclusion body washing buffer (100 mM sodium chloride, 1 mM EDTA, 1% Triton X-100, 2 M urea, 1 mM dithiothreitol, and 50 mM Tris-HCl, pH 8.0) and then solubilized in a dissolution buffer (8 M urea and 10 mM imidazole in phosphate buffer, pH 7.4). The soluble protein fraction and dissolved inclusion body proteins were purified with the HisPur Ni-NTA Purification Kit (88,229, Thermo, Germany). The purified inclusion body proteins were refolded by gradient dialysis in a dialysis refolding fluid. The expression and purification levels were analyzed by 15%SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and the protein content was determined with the BCA Protein Assay Kit (Thermo Fisher Scientific).
RGD4C penetration test
RGD4C penetrates different tumor cells
RGD4C-EGFP expressed in prokaryotes was used to trace RGD4C penetration of tumor cells. The human tumor cell lines U251, Huh7, SW480 and A549 with high integrin αvβ3 expression and the normal human lung epithelial cell line BEAS-2B were seeded in a 6-well plate at a cell density of 2 × 104, cultured in DMEM overnight and then cultured in DMEM containing RGD4C-EGFP. EGFP fluorescence was observed under an inverted fluorescence microscope.
Effect of an endocytosis inhibitor on membrane penetration
SW480 cells were seeded in 6-well plates and cultured overnight. After PBS washing, the endocytosis inhibitor chlorpromazine (50 μM), EIPA (50 μM) or MβCD (1 mM) was added to 300 μl of DMEM containing 10% FBS and co-incubated at 37 °C for 30 min. Then, the cells were incubated with 20 μM RGD4C-EGFP at 37 °C for 5 h. EGFP fluorescence was observed under an inverted fluorescence microscope.
Penetration time of RGD4C
SW480 cells were seeded one day in advance and cocultured with 20 μM RGD4C-EGFP at 37 °C in 0.5-h, 1-h, 2-h, and 5-h time gradients. Normal BEAS-2B cells were used as a control group. EGFP fluorescence was observed under an inverted fluorescence microscope.
Concentration dependence test
RGD4C-EGFP, at concentrations of 5 μM, 10 μM and 20 μM, was cocultured with previously seeded SW480 cells in 6-well plates for 5 h at 37 °C. EGFP fluorescence was observed under an inverted fluorescence microscope.
Temperature-dependent penetration test
SW480 cells were seeded one day in advance. 20 μM RGD4C-EGFP was added to the SW480 cells and incubated at 4 °C or 37 °C for 5 h. EGFP fluorescence was observed under an inverted fluorescence microscope.
Effect of ion concentration on membrane penetration
SW480 cells were treated with PBS (K+) in DMEM for 0.5 h, and then were cultured with 20 μM RGD4C-EGFP for 5 h. The control group was treated with PBS to detect the effect of extracellular potential differences on RGD4C peptide penetration. EGFP fluorescence was observed under an inverted fluorescence microscope.
Detection of the immunoreactivity of RGD4C-p21Ras-scFv
Western blot assay
Prokaryotically expressed K-p21Ras  was separated by SDS-PAGE, then transferred to polyvinylidene fluoride (PVDF) membranes and incubated with RGD4C-p21Ras-scFv. Next, the PVDF membranes were incubated with anti-Flag tag antibody (Abnova, #2368, China). Subsequently, the membranes were washed and incubated with a goat anti-mouse/rabbit IgG antibody and horseradish peroxidase (HRP) (ZSGB-Bio, ZB-5305, China) at 37 °C for 45 min. After washing with TBST, the protein bands were visualized with a 3,3′-diaminobenzidine (ZSGB-Bio) .
ELISA plates were coated overnight at 4 °C with 5 μg/ml K-p21Ras antigen in 0.05 M carbonate buffer at pH 9.6. The plates were then washed and blocked with 1% bovine serum albumin (BSA)-PBS at 37 °C for 1 h. RGD4C-scFv was diluted 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400 and 1:12800 with 10% BSA and then allowed to bind to the plates for 1 h at 37 °C. Other control proteins were treated in the same way. After incubation with anti-Flag tag monoclonal antibody (1:1000 dilution) for 1 h at 37 °C, the plates were subsequently incubated with an HRP-conjugated goat anti-mouse/rabbit detection antibody (ZSGB-Bio) (diluted 1:1000 in 10% BSA) for 1 h at 37 °C. Finally, the plates were processed using TMB (3,3′,5,5′-tetramethylbenzidine) peroxidase substrate system (Tiangen Biotechnology, Beijing, China). The absorbance was measured at 570 nm with a microplate reader (Bio-Rad, USA).
Tumor cell penetration test of RGD4C-p21Ras-scFv
Western blot analysis
SW480, Huh7, U251, and A549 tumor cells with high integrin expression and normal BEAS-2B cells without integrin expression were cultured with 20 μM RGD4C-scFv or RGD4C-linker-scFv for 5 h. The cells were lysed in RIPA lysis buffer with a protease inhibitor cocktail containing phenylmethylsulfonylfluoride (PMSF) for 30 min to extract total protein from the tumor cells. Then, the protein electrophoresis was performed with SDS-PAGE gels, and proteins were transferred to polyvinylidene fluoride (PVDF) membranes. β-actin was used as an internal control. Images were converted to the grayscale mode with Photoshop software. Quantification of the target proteins was accomplished by calculating the relative band intensity in the grayscale images of the proteins.
SW480 cells were cultured with 20 μM anti-p21Ras scFv, RGD4C-scFv, or RGD4C-linker-scFv. Then the cells were fixed in formalin, paraffin-embedded and sectioned. The sections were next exposed to a primary anti-Flag monoclonal antibody (Abnova, #2368, China) and secondary antibody at 1:3000 dilution. The DAB Detection Kit (ZSGB-Bio) was used for staining, and the slides were then counterstained.
SW480 cell lines were seeded on coverslips and cultured in dishes at 37 °C with 5% CO2, when 80% confluent cells were formed. 20 μM recombinant antibody RGD4C-scFv was added, and incubated for 5 h at 37 °C. And then fixed with 4% paraformaldehyde for 30 min. After permeabilized with PBS containing 0.2% Triton X-100 (Sigma-Aldrich, Darmstadt, Germany) and washed with PBS containing 0.02% Tween-20 (PBST)three-times, the slides were incubated overnight at 4 °C with primary rabbit anti-His Tag mAb (clone number: D3I1O, Cell Signaling TECHNOLOGY, USA) and mouse pan-Ras mAb (clone number: C4, SANTA CRUZ, USA), washed for 5 min with PBS then incubated for 1 h at 37 °C in the dark with FITC-conjugated goat anti-rabbit antibody (ZSGB-BIO) and TRITC-conjugated goat anti-mouse antibody (ZSGB-BIO). Nuclei were stained with 4′,6-diamidino-2-phenylindole (Sigma, Da, Germany) at 25 °C for approximately 15–20 min. The fluorescence signals were analyzed with a fluorescence microscope (OlympusBX51, Tokyo, Japan).
Antitumor activity of RGD4C-p21Ras-scFv in vitro
Cell migration assay
SW480 cells were cultured in 6-well plates to 80% confluence and then starved in serum-free medium overnight. Thereafter, the bottom of the culture plates was scratched with a 200-μl pipette tip. Then, 20 μM anti-p21Ras scFv, RGD4C-scFv, RGD4C-linker-scFv, RGD4C-scFv with 120 μM chloroquine (CQ) and RGD4C-EGFP were added, respectively. CACO-2 and CCD841 cells as the control group were cultured with same way. 20 μM RGD4C-scFv, RGD4C-scFv with 120 μM chloroquine, RGD4C-EGFP were added to the CACO-2 and CCD841 cells, respectively. Cell migration was detected under an inverted microscope (Olympus, Japan) at 0 h, 24 h, and 48 h, and the migration area was calculated using ImageJ software.
Colony formation analysis
SW480 cells were cocultured with anti-p21Ras scFv, RGD4C-scFv, RGD4C-linker-scFv, RGD4C-scFv with 120 μM chloroquine and RGD4C-EGFP respectively for 24 h. CACO-2 and CCD841 cells were cocultured with RGD4C-scFv, RGD4C-scFv with 120 μM chloroquine and RGD4C-EGFP, respectively. After digested with 0.25% trypsin and suspended in 10% FBS, the cells were cultured in DMEM containing 10% fetal bovine serum in 6-well plates for 2 weeks at 37 °C with 5% CO2. Cell growth was terminated when culture clones could be observed macroscopically. The cells were washed with PBS and fixed with methanol for 15 min. Following 1% Giemsa staining for 10–30 min, the cells were washed with water and dried in air. Colony-forming efficiency was calculated using the formula: colony-forming efficiency = (number of clones/inoculated cell count) × 100%.
Cell killing assay
SW480, CACO-2 and CCD841 cells at logarithmic growth phase were inoculated at a density of 1 × 104 cells per well in 96-well plates for 3 days, the anti-p21Ras scFv, RGD4C-scFv, RGD4C-linker-scFv, RGD4C-scFv with 120 μM chloroquine and RGD4C-EGFP were added to SW480 respectively, the RGD4C-scFv, RGD4C-scFv with 120 μM chloroquine, RGD4C-EGFP was added to CACO-2 and CCD841 respectively. At 1, 2, and 3 days, 20 μl of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (5 mg/ml) was added to each well. After 4 h of incubation with MTT, DMSO (100 μl/well) was added, and the plates were shaken for 10 min. The optical density (OD) value of each well was measured at 490 nm using a microplate reader (Bio-Rad, Model 680).
SW480 cells were treated with anti-p21Ras scFv, RGD4C-scFv, RGD4C-linker-scFv, RGD4C-scFv with 120 μM chloroquine and RGD4C-EGFP, respectively, the CACO-2 and CCD841 cells were treated with 20 μM RGD4C-scFv, RGD4C-scFv with 120 μM chloroquine, RGD4C-EGFP respectively for 10 h and then embedded in wax blocks for sectioning. Apoptosis was detected using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (In Situ Cell Death Detection Kit; Roche Diagnostics). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Apoptotic cells were visualized using a fluorescence microscope.
All data are presented as the mean value ± s.d. Each statistical analysis was performed using SPSS Version 22.0. Comparisons among all groups were performed with a one-way analysis of variance (ANOVA) and the Student–Newman–Keuls method. P values < 0.05 was considered statistically significant.