DOI: https://doi.org/10.21203/rs.3.rs-95670/v1
Background
An anti-p21Ras scFv can specifically bind with mutant and wild-type p21Ras but cannot penetrate the cell membrane, which prevents it from binding to p21Ras in the cytoplasm. Here, the RGD4C peptide was used to mediate scFv penetration into tumor cells and produce an inhibitory effect.
Methods
RGD4C-linker-EGFP and RGD4C-p21Ras-scFv recombinant expression plasmids were constructed, and the fusion proteins were expressed in E. coli and purified with HisPur Ni-NTA. RGD4C-linker-EFGP was used to test the factors affecting RGD4C penetration of the tumor cell membrane. The immunoreactivity of RGD4C-p21Ras-scFv toward p21Ras was identified by ELISA and western blotting. Moreover, immunocytochemistry was used to detect the ability of RGD4C-p21Ras-scFv to penetrate SW480 cells. Cell migration, colony formation, cell killing, and apoptosis assays were used to assess the inhibitory effect of RGD4C-p21Ras-scFv on SW480 cells in vitro.
Results
The RGD4C peptide could target tumor cells, but endocytosis inhibitors and a low temperature inhibited RGD4C peptide endocytosis into cells, and tumor cell entry was time and concentration dependent. Additionally, a change in the cell membrane potential did not affect penetrability. We found that RGD4C-p21Ras-scFv could penetrate SW480 cells; effectively inhibit the growth, proliferation and migration of SW480 cells; and promote apoptosis in SW480 cells.
Conclusion
The RGD4C peptide can mediate anti-p21Ras scFv entry into SW480 cells and produce an inhibitory effect, which indicates that RGD4C-p21Ras-scFv may be a potential therapeutic antibody for the treatment of RAS-mutant colorectal cancer.
Colorectal cancer (CRC) is the third most commonly diagnosed malignancy and one of the leading causes of cancer mortality worldwide and especially in China[1-3]. According to the relevant literature, colorectal cancer accounts for approximately 10% of all annually diagnosed cancers[4, 5]. Although surgery remains the only effective curative option for colorectal cancer[6], 50%-60% of tumors have metastasized when diagnosed, thus resulting in metastatic colorectal cancer (mCRC), which is incurable in most cases[7, 8]. In recent years, the application of targeted molecular drugs, such as bevacizumab, cetuximab and panitumumab[9], has led to a significant improvement in the survival rate of patients with metastatic colorectal cancer[10]. However, these targeted drugs also have certain limitations; for example, patients with metastatic colorectal cancer harboring mutations in exon 2 of K-ras do not benefit from anti-epidermal growth factor receptor (EGFR) therapy[11]; only those patients with wild-type ras genes benefit from such treatment [12]. Therefore, ras (K-ras/N-ras) mutation testing is highly recommended in the National Comprehensive Cancer Network (NCCN) guidelines and other guidelines [13-15].
The ras (H-ras, K-ras, and N-ras) family encodes a group of 21,000-dalton proteins (p21Ras) and includes the most commonly mutated genes in all human malignancies, including colon cancer [16-18]. According to a report, K-ras mutations are present in 22% of tumors, while N-ras and H-ras mutations are less frequent at 8% and 3%, respectively[19, 20]. Ras gene mutations occur in over a third of human colorectal cancer cases[21], and the mutation rate of K-ras is as high as 30% - 60%[22-25]. Mutated p21Ras proteins become key drivers in the development of cancers[26, 27]. Moreover, wild-type p21Ras overexpression is an important cause of colorectal cancer [28], and the expression rate of p21Ras has been found to reach 29% - 76%[29]. p21Ras could become a promising therapeutic target for colorectal cancer treatment. To date, however, there is currently no effective and safe treatment to directly target ras-driven neoplasms. Therefore, it is necessary to develop a novel high-efficiency drug that can inhibit mutant p21Ras and overexpressed wild-type p21Ras.
To block the ras signaling pathway and target tumors driven by the ras gene, we previously constructed a single-chain variable fragment antibody (scFv) against p21Ras (anti-p21Ras scFv)that can specifically bind to both mutant p21Ras and wild-type p21Ras[30]. However, the anti-p21Ras scFv cannot penetrate the cell membrane, which prevents it from binding to p21Ras in the cytoplasm. As a consequence, it is vital to select an alternative vector to carry the anti-p21Ras scFv into tumor cells to exert antitumor effects.
"Cell-penetrating peptides" (CPPs) are natural or synthetic peptides with the ability to interact with cell membranes to enter cells and/or deliver cargo[31]. Currently, CPPs have been widely used as carriers for the delivery of macromolecular drugs, not only enhancing intracellular drug delivery but also improving targeting[32]. RGD, an arginine-glycine-aspartic acid tripeptide, is the interacting site between an integrin and its ligand and shows binding to a variety of integrins[33, 34]. Integrins and RGD-based ligands for integrins are currently being investigated in drug delivery-related areas of research[35, 36]. Alpha(v)beta(3) (αvβ3) is an important integrin. Previous studies have reported that αvβ3 is specifically overexpressed in activated endothelial cells and tumor cells but is not expressed or is rarely expressed in the vast majority of mature endothelial cells and normal cells[37, 38]. Thus, the integrin αvβ3 could become a promising target for cancer therapy. According to these characteristics, several peptides containing RGD sequence-based delivery systems have been designed to specifically bind to αvβ3 receptors. These receptors not only improve targeting potential but also enhance cell membrane internalization to allow therapeutic drugs to enter tumor cells [39, 40]. Assa-Munt N. et al originally isolated the RGD4C peptide (ACDCRGDCFCG) from a phage-displayed peptide library by screening with the αvβ5 integrin[41]. RGD4C contains the RGD sequence, which can avidly bind to the integrins αvβ3 and αvβ5 but does not bind to other closely related integrins[42]. In addition, the RGD4C peptide can enhance tumor uptake and enable selective delivery of therapeutic or diagnostic agents to tumor sites[43, 44]. In this study, to improve the penetration of the anti-p21Ras scFv into tumor tissues via endocytosis, we connected the RGD4C sequence to the N terminus of the anti-p21Ras scFv to construct RGD4C-p21Ras-scFv prokaryotic expression vectors. We then expressed and purified the fusion protein RGD4C-p21Ras-scFv (RGD4C-scFv and RGD4C-linker-scFv) and subsequently investigated the effects on targeting and penetrating the human colorectal cancer cell line SW480 as well as the antitumor effect in vitro.
Cell line and culture
The human non-small cell lung cancer cell line A549, human colon cancer cell line SW480, human hepatoma cell line Huh7, human glioma cell line U251 and normal human lung epithelial cell line BEAS-2B 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 ug/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[30]. The RGD4C peptide (ACDCRGDCFCG) was developed with phage display technology[45]. 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-linker-EGFP.
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 inducibly expressed with 1 mM isopropyl-β-d thiogalactoside (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 (88229, Thermo, Germany). After the inclusion body proteins were denatured in urea, the 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 penetrates different tumor cells
RGD4C-linker-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-linker-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 coincubated at 37 ℃ for 30 min. Then, the cells were incubated with 20 µM RGD4C-linker-EGFP at 37°C for 5 h. EGFP fluorescence was observed under an inverted fluorescence microscope.
Penetration time test
SW480 cells were seeded one day in advance and cocultured with 20 µM RGD4C-linker-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-linker-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℃. EGFP fluorescence was observed under an inverted fluorescence microscope.
Temperature-dependent penetration test
The effect of temperature on the penetration efficiency of RGD4C-linker-EGFP was tested at 4°C and 37°C SW480 cells were seeded one day in advance, and 20 µM RGD-linker-EGFP was added to the SW480 cells and incubated at 4℃ or 37℃ 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 the SW480 cells were cultured with 20 µM RGD4C-linker-EGFP for 5 h. In contrast, 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.
Western blot assay. Prokaryotically expressed K-p21Ras[46] was separated by SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes and incubated with RGD4C-p21Ras-scFv. Next, the PVDF membranes were incubated with a primary anti-Flag tag antibody (Abnova, #2368, China) after blocking. 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 chemiluminescence system (ZSGB-Bio) [29].
ELISA. 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 a mouse 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 a 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).
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, electrophoresis was performed with SDS-PAGE gels, and proteins were transferred to polyvinylidene fluoride (PVDF) membranes. The experimental method was performed as described previously. 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.
Immunocytochemical staining. SW480 cells were cultured with 20 µM anti-p21Ras scFv, RGD4C-scFv, or RGD4C-linker-scFv. Then, they 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 a 1:3000 dilution for staining. The DAB Detection Kit (ZSGB-Bio) was used, and the slides were then counterstained.
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, followed by washing three times with PBS. Then, 20 µM anti-p21Ras scFv, RGD4C-scFv or RGD4C-linker-scFv was added to the cells; PBS was used in the negative control group. 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 or RGD4C-linker-scFv for 24 hours. Then, the tumor cells were digested with 0.25% trypsin and suspended in 20% FBS. The cells were cultured in DMEM containing 20% fetal bovine serum in 6-well plates for 2 weeks at 37°C with 5% CO2. Cell line 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) x 100%.
Cell killing assay. Logarithmic growth phase cells were inoculated at a density of 1 × 104 cells per well in 96-well plates. After 3 days, anti-p21Ras scFv, RGD4C-scFv, or RGD4C-linker-scFv was added to the cells, and the control group was treated with PBS. 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).
Apoptosis assay. SW480 cells were treated with 20 µM RGD4C-scFv for 5 h and then embedded in wax blocks for sectioning. Apoptotic cells were 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 SW480 cells were visualized using a fluorescence microscope.
Statistical analysis
All date 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.
The construction of prokaryotic recombinant expression plasmids is shown in Figure 1A. An E. coli expression system was used to prepare all the fusion proteins. PCR showed that the recombinant plasmids containing the target gene fragments were successfully transformed into E. coli BL21 (DE3) (Figure 1B). The molecular weight of fusion proteins was determined by SDS-PAGE after purification with nickel metal-affinity resin columns. All of the fusion proteins matched the expected molecular weight, which was 34 kDa for anti-p21Ras scFv, 35 kDa for RGD4C-scFv, 36 kDa for RGD4C-linker-scFv, and 34 kDa for RGD4C-linker-EGFP, and no degradation was observed (Figure 1C). A BCA assay showed that the concentration of purified anti-p21Ras scFv, which was not codon optimized, was 0.96 mg/ml, and that of RGD4C-linker-scFv was 1.06 mg/ml. However, the concentration of codon-optimized anti-p21Ras scFv was 1.41 mg/ml, that of RGD4C-scFv was 1.34 mg/ml, and that of RGD4C-linker-scFv was 1.27 mg/ml. Therefore, the results revealed that fusion protein expression was higher after codon optimization.
The capacity of the RGD4C peptide to mediate penetration of tumor cells with integrin αvβ3 expression was assessed. Fluorescence microscopy was used to observe whether RGD4C-linker-EGFP entered tumor cells with high integrin expression. The green fluorescence signal of RGD4C-linker-EGFP was found in tumor cells, but no fluorescence signal was found in the normal cell line BEAS-2B. These results indicated that RGD4C could penetrate tumor cells with integrin expression but could not penetrate normal cells (Figure 2A).
Different factors affecting the penetration of the RGD4C peptide into tumor cells were evaluated. When different endocytosis inhibitors were added to cocultures of RGD4C-linker-EGFP and SW480 cells, strong fluorescence was observed in the PBS control group, but weak fluorescence was observed in the inhibitor groups (Figure 2B and G). After RGD4C-linker-EGFP was added to SW480 cells, the green fluorescence signal increased as the culture time increased, and the fluorescence signal was strongest at 5 h (Figure 2C). When the RGD4C-linker-EGFP concentration was 20 µM, the green fluorescence observed was significantly stronger than that in other experimental groups (Figure 2D). In an incubation temperature test, stronger green fluorescence was observed at 37°C, which indicated that the low temperature of 4°C could inhibit the penetration efficiency of the RGD4C peptide (Figure 2E and H). Furthermore, the effect of cell membrane potential on the penetration of RGD4C peptide was analyzed and showed that changing cell membrane potential did not affect RGD4C entry into SW480 cells (Figure 2F and I).
RGD4C-p21Ras-scFv immunoreactivity was analyzed by western blotting and ELISA to determine whether the RGD4C peptide affects the biological activity of the anti-p21Ras scFv. Western blotting showed that the anti-p21Ras scFv, RGD4C-scFv and RGD4C-linker-scFv could interact with the K-p21Ras antigen, implying that the RGD4C peptide and linker peptide had no effect on the immune activity of the scFv. ELISA results revealed that the binding titers of RGD4C-scFv and RGD4C-linker-scFv for the p21Ras antigen were 1:800, similar to the titer of the anti-p21Ras scFv, which further confirmed that the RGD4C peptide and linker peptide did not affect the titer of the anti-p21Ras scFv (Figure 3A).
The effects of RGD4C-scFv and RGD4C-linker-scFv penetration on SW480 cells were evaluated by immunocytochemistry. Immunocytochemical staining analysis showed that there were high levels of RGD4C-scFv and RGD4C-linker-scFv in SW480 cells, but no positive cells were found in the control anti-p21Ras scFv and PBS groups (Figure 3B). Moreover, western blotting revealed that RGD4C-scFv and RGD4C-linker-scFv were detected in tumor cells with high integrin expression, but no fusion proteins were detected in normal cells. RGD4C-scFv and RGD4C-linker-scFv had the same targeted penetration ability as RGD4C, and they could penetrate all the tested tumor cell membranes and enter tumor cells. Overall, the results showed that the RGD4C peptide could guide the anti-p21Ras scFv to penetrate tumor cells with high expression of integrin αvβ3. At the same time, the linker did not affect the ability of the RGD4C peptide to carry the scFv into tumor cells (Figure 3C).
A scratch test revealed that the area of migrating cells was significantly larger in the anti-p21Ras scFv and PBS treatment groups than in the RGD4C-scFv and RGD4C-linker-scFv treatment groups (Figure 4A-B), suggesting that RGD4C-scFv and RGD4C-linker-scFv can inhibit the migration of SW480 cells.
Consistently, the clone formation rates of SW480 cells were 29.58 7.89% in the RGD4C-scFv treatment group and 31.00 7.85% in the RGD4C-linker-scFv group but 70.92 10.42% in the anti-p21Ras scFv group and 75.17 16.50% in the PBS group. This colony formation assay demonstrated that RGD4C-p21Ras-scFv fusion proteins could inhibit the proliferation of SW480 cells (Figure 4C).
An MTT assay was performed to evaluate the killing effects of RGD4C-scFv and RGD4C-linker-scFv on SW480 cells. We found that the numbers of live tumor cells in the RGD4C-scFv and RGD4C-linker-scFv groups were lower than those in the anti-p21Ras scFv and PBS groups (Figure 4D).
TUNEL analysis demonstrated that apoptotic cell numbers increased significantly after treatment with RGD4C-scFv or RGD4C-linker-scFv compared with control treatment. The percentages of apoptotic cells were 54.6 12.1% in the RGD4C-scFv group and 51.6 8.5% in the RGD4C-linker-scFv group. However, in the anti-p21Ras scFv group, the percentage of apoptotic cells was 12.2 2.3%, and there was a significant difference between the RGD4C-scFv and anti-p21Ras scFv groups (P < 0.01) (Figure 4E -F). Taken together, the above results indicate that RGD4C can carry the anti-p21Ras scFv into SW480 tumor cells and that RGD4C-p21Ras-scFv has antitumor activity in vitro.
In the present study, we chose the common cell-penetrating peptide RGD4C as the guiding peptide to carry the anti-p21Ras scFv into tumor cells and constructed a prokaryotic expression system for recombinant RGD4C-p21Ras-scFv fusion proteins in vitro. Then, we assessed the factors affecting penetration of the cell membrane by RGD4C and the antitumor activity of the fusion proteins against human colon cancer cells. Additionally, we evaluated whether the linker protein between the RGD4C peptide and anti-p21Ras scFv could influence the biological activity of the RGD4C-p21Ras-scFv fusion protein.
As a guide peptide, the RGD4C peptide has the ability to carry macromolecular drugs through membranes. In recent years, many studies have suggested that the addition of an RGD fragment to peptide drugs may solve the serious limitation of numerous antitumor drugs being unable to penetrate solid tumors. Natasa Zarovni et al. used the RGD4C peptide to carry tumor necrosis factor alpha (TNF), and their experiments showed that the RGD4C peptide successfully increased the uptake of an antibody specific for a tumor-associated antigen and improved the therapeutic properties of the TNF gene[47]. Ebrahim Hosseini et al. showed that modification of interleukin‑24 (IL‑24) with RGD4C fragments enhanced adherence to tumor cells and improved the anticancer activity of interleukin‑24 (IL‑24)[48]. Furthermore, some studies have also indicated that the RGD peptide is internalized into endosomal compartments by binding to αvβ3 receptors. In this study, we conjugated the RGD4C peptide to the anti-p21Ras scFv to improve the ability of the anti-p21Ras scFv to penetrate SW480 tumor cells in a targeted manner. Fortunately, in our transmembrane experiment, the results showed that the RGD4C peptide included in fluorescent protein conjugates could induce targeted endocytosis to cross the tumor cell membrane, and immunocytochemistry results showed that RGD4C-p21Ras-scFv fusion proteins were able to target and accumulate in SW480 cells because the fusion proteins were powerfully recognized and internalized by integrin αvβ3 receptors expressed on the SW480 tumor cells. Nevertheless, the anti-p21Ras-scFv was not detected in SW480 cells, which was consistent with the expected results.
Because the molecular weight of the EGFP protein is similar to that of the anti-p21Ras scFv, we labeled the RGD4C peptide with EGFP to create an RGD-linker-EGFP fusion protein identifiable by green fluorescence after expression. In the tumor-targeting experiment with the RGD4C peptide, green fluorescence existed in tumor cells with high integrin expression but not in normal cells, which indicated that the RGD4C peptide could cross the cell membrane by recognizing the integrin αvβ3 on the surface of the tumor cells. When changes were made in concentration, temperature, time, endocytosis inhibition or the potential difference, the RGD4C peptide was found to have concentration- and time-dependent membrane penetrating effects. It was found that the penetration ability of the RGD4C peptide was weakened after endocytosis inhibitor addition, which indicated that RGD4C function through an endocytosis mode. Moreover, the cell membrane potential did not affect RGD4C entry into tumor cells, suggesting that RGD4C peptide entry into tumor cells occurs via energy-independent endocytosis.
During the construction and expression of fusion proteins, to ensure the activity and function of two interconnected proteins, a specific protein linker may need to be added between the different proteins in the fusion protein to maintain the functions of the various proteins. In our study, we designed two kinds of RGD4C-p21Ras-scFv fusion proteins, and the difference between the two fusion proteins was whether they contained a linker binding peptide. In comparisons of the two fusion proteins, our data demonstrated that the absence of the linker protein did not affect immunoreactivity or the endocytosis pathway. In addition, there was no significant difference in the antitumor effect between the two fusion proteins in vitro. Therefore, we chose RGD4C-p21Ras-scFv, a simple fusion protein without the linker protein, for subsequent analysis of antitumor efficacy in vivo.
Our current study suggested that the RGD4C peptide could mediate anti-p21Ras scFv targeting to penetrate tumor cells and produce antitumor effects. In vitro, RGD4C-p21Ras-scFv fusion proteins inhibited the migration and proliferation of SW480 cells and induced apoptosis in SW480 cells. As a result, these characteristics of RGD4C-p21Ras-scFv fusion proteins could provide the premise for anti-colorectal cancer efficacy in vivo. Similarly, all the results suggest that the RGD-p21Ras-scFv conjugate can be used as a novel candidate targeted antitumor drug for colorectal cancer therapy. However, future studies are needed to assess the following: (a) the in vivo antitumor activity of RGD4C-p21Ras-scFv fusion proteins against human cancers; (b) the stability of RGD4C-p21Ras-scFv fusion proteins in the human body; and (c) immunogenicity and toxicity.
In summary, our experimental results confirmed that a prokaryotic expression system was successfully used to express recombinant RGD4C-p21Ras-scFv fusion proteins. The fusion proteins can enter into tumor cells, and showing a significant inhibitory effect against the growth of SW480 cells in vitro. Accordingly, the established therapeutic strategy of using RGD4C peptide carry anti-p21Ras scFv entry into tumor cells is feasible and effective. In future studies, we hope to develop the RGD4C-p21Ras-scFv as a potential therapeutic antibody for the treatment of ras-mutant colorectal cancer and other tumors with overexpressed p21Ras and ras mutations.
CRC: Colorectal cancer; mCRC: metastatic colorectal cancer; EGFR: epidermal growth factor receptor; scFv: single-chain variable fragment antibody; CPPs: Cell-penetrating peptides; HRP: Horseradish peroxidase; IHC: Immunohistochemistry; MTT: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; PVDF: Polyvinylidene fluoride;
Ethics approval and consent to participate
No cell lines used in the work presented in this paper required Ethics approval.
Consent for publication
Not applicable.
Availability of data and materials
The data and materials used and analyzed in the current study would be available from the corresponding author on request.
Competing interests
The authors declare that they have no competing interests.
Funding
The Applied Foundation Key Project of Yunnan Province (2018ZF009) and the National Natural Science Foundation of China (No. 81460464). The funding body was not directly involved in the design of the study or collection, analysis, and interpretation of data or in writing the manuscript.
Authors’ contributions
CCH performed manuscript writing and organized the data for the manuscript, and FRL carried out most of the experiments. QF and XYP participated in the data organization and manuscript drafting. SLS performed the immunohistochemistry. JLY designed the project. All authors read and approved the final manuscript.
Acknowledgements
This work was supported by grants from the Applied Foundation Key Project of Yunnan Province(2018ZF009) and the National Natural Science Foundation of China (No. 81460464).