Background
Melanoma, which can be cured by surgery in the early stage but the prognosis is poor, accounts for about 80% of skin cancer deaths. This is largely because metastatic malignancy is refractory to conventional therapies [
1‐
4]. Gene mutations, mostly BRAF mutations, are present in over half of all human melanomas. Although patients with BRAF-mutant are highly sensitive to small molecule inhibitors, like Vemurafenib and Dabrafenib, most patients display partial responses to clinical treatment, which can eventually worsen conditions or cause death due to drug resistance within 6 to 12 months [
5‐
7]. Moreover, patients that lack gene mutations have few effective therapeutic methods. These patients are associated with unique risk factors, pathophysiologic, clinical, and prognostic features that differ from those related to BRAF tumors [
8]. Thus, it is necessary and urgent to address the problems of drug resistance to inhibitors with BRAF-mutant and lacking appropriate therapies for patients without specific mutations.
Src Homology 2 (SH2) domains specifically recognize phosphorylated tyrosine and mediate cell signal transduction [
9,
10]. The “superbinder” SH2 domain, with triple-point mutants Thr8Val/Cys10Ala/Lys15Leu, abbreviated SH2 superbinder, which could bind pY-containing peptides with much stronger affinity than natural SH2 domains or traditional anti-pY antibody (4G10) [
11,
12]. Conventional drugs are generally directed against a single gene or protein and most of melanoma patients develop drug resistance eventually [
13]. SH2 superbinder, strongly binding with diverse pY sites to block related signal transduction pathways, can recognize other sites instead, even if mutation alters the pY site [
14,
15]. Therefore, SH2 superbinder can achieve the purpose of killing cancer cells [
16]. These advantages might resolve problems of drug resistance due to gene mutation and lack of suitable targeting drugs described above. However, it is urgent to settle the problem of transporting SH2 superbinder across cell membrane barriers.
Cell penetrating peptides (CPPs), having ability to translocate across the plasma membranes, are confined to short sequences of less than 20 amino acids [
17]. CPPs have several advantages over conventional techniques on cellular delivery because they are efficient for a variety of cells, and have a potential therapeutic application [
18]. Nona-arginine (Arg)
9, as one kind of CPPs, has been applied efficaciously to translocate across the cell membrane and deliver large cargo molecules such as peptides, proteins, and oligonucleotides into cells with no severe side effects, which has a prospect of wide application and a development potential in drug delivery [
19,
20].
(Arg)9-SH2 superbinder (referred as (Arg)9-GST SH2 TrM) was conducted by genetic engineering approach. The results validated that (Arg)9-SH2 superbinder had strong ability to translocate into cells and played a role in melanoma cells by binding with various pY proteins. Furthermore, (Arg)9-SH2 superbinder could suppress proliferation and migration of melanoma cells. Meanwhile, apoptosis caused by (Arg)9-SH2 superbinder was observed. Additionally, PI3K/AKT, MAPK/ERK and JAK/STAT pathways were also affected by (Arg)9-SH2 superbinder. Moreover, (Arg)9-SH2 superbinder could inhibit the growth of tumor in vivo. Above all, (Arg)9-SH2 superbinder, which could translocate into cells efficaciously and provide a novel anticancer method for melanoma via capturing multiple pY-containing proteins, might be a potent candidate in the targeted therapy of melanoma.
Methods
Construction, expression and purification of GST fusion proteins
Genes encoding the human wild type and triple-mutant Src SH2 domains, from the pEGFP-C3-Src SH2 Wt/TrM plasmids, were subcloned into the pGEX-4 T3 vector for expressing GST-Src SH2 Wt/TrM proteins. Based on pGEX-4 T3-alone or pGEX-4 T3- Src-SH2 Wt/TrM, recombinant plasmids were constructed for expressing GST-(Arg)
9, GST-Src-Wt/TrM-(Arg)
9. Wt represented Wild type. Amino acid sequences and primers of all used constructs were listed in Additional file
1: Table S1 and Additional file
2: Table S2 of supplementary materials. As shown in Additional file
1: Table S1, although nona-arginines were put at the end of C-terminus of GST or GST-Src-SH2 Wt/TrM, we termed them as (Arg)
9-GST or (Arg)
9-GST-Src-SH2 Wt/TrM.
All amplified products were gel purified using the Gel Band Purification Kit (Beijing ComWin Biotech). The backbone was digested with BamHI (cat. # R3136V) and NotI (cat. # R3189V) in 37 °C for 2 h and the purified PCR products were reacted with the linearized pGEX-4 T3 vector at 37 °C for 30 min. The experiment is using the One Step Cloning Kit (Vazyme, Nanjing), according to the principle of homologous recombination. E.coli BL21 containing the expression plasmid was grown in LB broth with 100 μg/ml ampicillin at 37 °C. The expression of GST fusion protein was induced by the addition of isopropyl β-D-thiogalactoside (0.5 mM final concentration), and then incubated at 20 °C for 18 h. The lysis buffer of protein contains 20 mM Tris-HCl (pH 7.0), 50 mM NaCl, 0.5 mM EDTA, 1 mM dithiothreitol (DTT), 1 mM cocktail, and 1 mM PMSF. GST fusion proteins were purified from bacterial cell lysates by glutathione-agarose beads. After sonication, cell lysates were cleared by centrifugation at 9500 rpm for 30 min, prior to mixing with glutathione-agarose beads. After rotating at 4 °C for 3 h, proteins could be eluted and collected. The protein concentration in the cell homogenates was quantified with BCA Protein Assay Kit. Immediately prior to their use in biological assays, protein purity was verified by SDS-PAGE using Coomassie brilliant blue staining intensity.
Cell lines and cell culture
B16F10 melanoma cells (no metastasis variant mouse melanoma), A375 (BRAF mutation) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). The cisplatin (DDP)-resistant subline A375/DDP was established with continuous exposure of the parental A375 cells to increasing concentrations of cisplatin, ranging from 2 nM to 4 μM for about 6 months. The drug-resistant cells were maintained in DMEM containing 4 μM cisplatin. All cells were cultured in DMEM medium supplemented with 10% FBS and 100 U/mL penicillin- streptomycin and were maintained in a humid atmosphere with 5% CO2 at 37 °C.
Glutathione s-transferase pull down assay and western blot
For GST pull down assay, GST fusion proteins were expressed in E. coli BL21 (DE3). Cells were treated with phosphatase inhibitor sodium pervanadate (0.5 mM) for 10 min at 37 °C before harvesting. Then, cells were lysed in ice-cold lysis buffer (0.5% NP-40, 50 mM Hepes (pH 7.4), 1 mM magnesium chloride, 150 mM KCl, and the complete protease inhibitor cocktail). For immunoprecipitation and western blot (immunoblot), cells were lysed on ice in lysis buffer (1% NP-40, 50 Mm Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 10% glycerol, and the complete protease inhibitor cocktail). The supernatant was gathered after centrifugation at 12,000 g for 15 min. Protein A/G agarose (Thermo Fisher) and Glutathione Sepharose beads (GE Healthcare) were used for the immunoprecipitation and GST pull down assays, respectively. Protein concentrations were quantified by BCA method. The proteins were separated by a 10% SDS-polyacrylamide gel and eleco-transferred onto PVDF membranes (Millipore), which were incubated in 5% skim milk for 1 h at room temperature. Primary antibodies against EGFR(CST#4267), Grb2(CST#3972), pERK1/2(CST#4370), pSTAT3(CST#4113), pAKT(CST#4060), AKT(CST#9272), ERK1/2(CST#4695), STAT3(CST#4904), pY antibody (Abcam EPR16871), GAPDH(CST#5174), Bax(ABclonal#A12009) and Bcl2(ABclonal#A11025) were diluted at 1:1000 and then incubated with the membranes overnight at 4 °C. Membranes were washed three times for 10 min and incubated with a 1:5000 dilution of HRP-conjugated anti-mouse or anti-rabbit antibodies. Blots were washed with TBST three times and developed with the ECL system; the membranes were exposed to ChemiDoc MP Imager (BIO-RAD). The band densities were normalized relative to the relevant GAPDH with Image J software.
Immunofluorescence
1 × 104 cells were seeded in a 12-well plate and cultured for 24 h. Cells were incubated with proteins with different time and concentrations. After washing with cold washing buffer, cells were then fixed in 4% formaldehyde at room temperature for 1 h, and then were permeabilized with 0.5% Triton X-100 for 20 min. After rinsing in PBS, cells were treated with rhodamine phalloidin for 30 min and then incubated with DAPI for 5 min at RT. Samples were imaged by a fluorescence microscope (Olympus, Japan). The images were analyzed by Image J software.
MTT assay
Cells collected in the logarithmic phase were plated into 96-well plates (3–5 × 103 cells/well). On the following day, add GST fusion proteins into the cell culture medium. After incubating for different time, 10 μL of 5 g/L MTT solutions (Sigma) were added into each well and incubated for 4 h, and then incubated with 100 μL DMSO for another 15 min. The optical absorbance was measured at the wavelength of 570 nm. Different treatment time and concentrations have been shown in the figure legends.
CCK-8 assay
Cells collected in the logarithmic phase were plated into 96-well plates (5 × 103 cells/well). On the following day, add GST fusion proteins into cell culture medium with different concentrations. After incubating for various time, 10 μl CCK-8 solution (Dojindo) was added into each well and incubated for 1-4 h. The optical absorbance was measured at the wavelength of 450 nm.
Cells were trypsinized and plated in 6-well plates (100 cells/well), incubated with GST fusion proteins and counted 14 days after seeding. The colonies were subsequently fixed with 4% formaldehyde and stained with 0.01% crystal violet for 10 min.
Wound healing assay
Cells were seeded into six-well plates (5 × 105 cells/well). Wounds were then created using the 200 μL pipette tips. The scratched cells were removed by PBS for three times. Cells were then cultured for 16 h incubated in the presence or absence of (Arg)9-GST SH2 TrM. Microscopic images were taken with a digital camera at different time points.
Transwell assay
2 × 104 cells were plated in 200 μL DMEM containing 2% FBS in the upper chamber. The lower chamber was filled with 500 μL completed medium containing 10%FBS. (Arg)9-GST SH2 TrM or (Arg)9-GST were added to the upper chamber and cells were allowed to migrate for 16 h at 37 °C with 5% CO2. Cells were fixed with 4% formaldehyde for 15 min at room temperature. Then, cells on the upper chamber were moved with a cotton swab. After washing the chambers with ddH2O, cells remained on the bottom of the lower chamber were stained with 0.1% crystal violet. The migrated clones were photographed under a microscope. The cell numbers were counted at three different areas.
Apoptosis analysis
Cells were sedimented by centrifugation, resuspended and fixed in 100 μl binding buffer. Cell density in the cell suspension was adjusted to 2 × 103cells/μl. Subsequently, 5 μl Annexin V-FITC was added to the cell suspension followed by gently vortexing and incubation for 10 min at room temperature in the dark. Thereafter, the cell suspension was incubated with 5 μl Propidiumiodide. All details need to refer to the instructions of kit (Tianjin Sungene Biotech). Cells were analyzed immediately using a FACS flow cytometer (FACS BD Biosciences, Germany) for Annexin V-FITC and Propidiumiodide binding. Dot plots and histograms were analyzed by Flowjo software.
TdT-UTP nick end labeling (TUNEL) assays were performed with one-step TUNEL apoptosis assay kit produced by Beyotime Institute of Biotechnology according to the manufacturer’s instructions. The FITC-labeled TUNEL-positive cells were imaged under a fluorescent microscope (Olympus, Japan). The cells with green fluorescence were defined as apoptotic cells. And images were analyzed by Image J software.
Animal studies
Xenograft model was established on C57Bl/6 mice (Animal Center of Tongji Medical College, Wuhan), 4–6 weeks old, weighing approximately 18-22 g. All studies involving animals were performed following the National Guides for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Tongji Medical College, Huazhong University of Science and Technology.
A suspension of 1 × 106 B16F10 cells (in 100 μl PBS) was subcutaneously injected into the right flank each mouse. After the development of an easily palpable tumor (7–10 days, approximately 5 mm in diameter). Tumor size was measured by using a caliper every 3 days and was calculated by using the following formula: tumor volume = 1/2(width)2 × length. Mice were divided into control group (B16F10 cells and PBS injected) and the experimental group (B16F10 cells and (Arg)9-GST SH2 TrM treated), n = 3 per group. Animals were euthanized when tumor size reached the ethical end point.
Preparation of PBMCs
Peripheral blood obtained from healthy C57Bl/6 mice was anticoagulated with heparin. PBMC were isolated by Ficoll (Germany) density gradient centrifugation of peripheral venous blood. Cells were washed three times and resuspended in DMEM medium supplemented with 1 mM sodium pyruvate, 1% nonessential amino acids and vitamins, 2 mM L-glutamine, 100 U/ml penicillin-streptomycin and 10% FBS.
Statistical analysis
The Student’s test was used to test for statistical significance of the differences between the different group parameters. P values of less than 0.05 were considered statistically significant.
Discussion
Patients, with BRAF (V600) in 40–60% of melanomas, can be successfully cured with selective inhibitors, bringing about significant prolongation of progression free survival and overall survival [
35,
36]. Although BRAF inhibitors are effective treatment methods, drug resistance is inevitable, and high drug prices increase the burden on patients [
37‐
39]. Meanwhile, the remaining 50–60% of patients are BRAF wild type and they do not benefit from treatment with BRAF inhibitors. A number of other mutations are present in these patients, such as NRAS, MEK1/2 and atypical (non-V600) BRAF mutations [
8]. Treatment of this subset of patients without a BRAFV600 mutation is a challenging problem.
EGF, an extracellular signal factor, can activate downstream Ras, by binding with the EGFR, and then phosphorylate Raf, decreasing Ras-GTPase activity. The EGF-dependent signal pathways are closely related with tumorigenesis [
40]. BRAF, an important member of Raf family, is the downstream effector of RAS. Protein tyrosine phosphorylation plays an essential role in the development and progression of cancer. Aberrant tyrosine phosphorylation is associated with cancer [
41,
42]; Carcinogenic cells tend to exhibit abnormal high levels of phosphorylated tyrosine [
43,
44]. Previous studies have proved that the inactivation of MAPK/ERK signaling pathways can regulate migration of HEK293T cells [
16]. The tight combination of SH2 superbinder and EGFR could block the activation of downstream signaling pathways [
16]. Similarly, our results substantiated that levels of pAKT, pERK1/2 and pSTAT3 were reduced by (Arg)
9-SH2 superbinder via blocking the EGFR signaling pathway. Remarkably, the effects of (Arg)
9-SH2 superbinder on pathways described above were involved in the regulation of cell apoptosis. There are a large number of members in Bcl-2 family involved in regulation of apoptosis, such as Bcl-2, Bcl-X1, Bcl-w, Bax, Bad, Bak, Bcl-xs, Mc1–1 et al. Bcl-2 and Bax, two most popular members of this family, were chosen to be examined in this study. The results indicated pro-apoptotic protein Bax increased and anti-apoptosis protein Bcl-2 declined in B16F10 cells after (Arg)
9-SH2 superbinder incubation. Of course, it needs further investigations to check whether other members in Bcl-2 family mediate the process of (Arg)
9-SH2 superbinder-induced apoptosis in future.
In addition, the powerful vector (Arg)
9, for the intracellular delivery of conjugated large molecules, was applied efficaciously to translocate across the cell membrane and transport SH2 superbinder into the cytoplasm or nucleus of B16F10 cells. The results showed that fluorescence intensity of cells incubated with (Arg)
9-GST SH2 TrM was higher than that of (Arg)
9-GST because of the strong affinity between (Arg)
9-GST SH2 TrM and pY-containing proteins in cells. Indeed, cell penetrating peptides inevitably have some toxicity effect on the cells. The MTT data in our work showed that (Arg)
9-GST protein exhibited remarkably low toxicity, which is consistent with previous reports [
45,
46]. In brief, (Arg)
9 overcame the challenge of transporting therapeutic agents across cell membrane which showed a great value of application in biomedical system. Furthermore, we isolated the peripheral blood mononuclear cells(PBMCs) from mice and evaluated the penetration and toxicity effect of (Arg)
9 fused proteins on PBMCs. As shown in Additional file
8: Figure S6a, the level of pY was very low in PBMCs as the non-cancerous cells. (Arg)
9-GST-SH2 TrM protein could enter into PBMCs, but the green signal was very weak (Additional file
8: Figure S6b). Indeed, (Arg)
9-GST-SH2 TrM protein caused some toxicity effect on PBMCs according to the CCK-8 data (Additional file
9: Figure S7), but it was moderate as (Arg)
9-GST-SH2 TrM protein specifically recognizing and binding to pY residues, whereas the level of pY residues was verified to be very low in PBMCs.
These findings suggested that (Arg)9-SH2 superbinder could play a significant role in the progression of cell proliferation, migration and apoptosis. Importantly, these data implied that (Arg)9-SH2 superbinder, targeting multiple pY proteins, might be a useful anticancer therapy to address the difficulties of resistance and provide a novel effective way for melanoma.