|Year : 2022 | Volume
| Issue : 2 | Page : 352-361
Synergistic effect of growth factor receptor-bound protein 2/epidermal growth factor receptor dual-targeting peptide inhibitor and salinomycin on osteosarcoma
Zuochong Yu1, Yanlong Xu2, Longhai Du2, Fan Zhang3, Minghao Shao3, Lin Xie3, Guoping Cai2, Feizhou Lyu3
1 Department of Orthopedics, Huashan Hospital of Fudan University; Department of Orthopedics, Jinshan Hospital of Fudan University, Shanghai, China
2 Department of Orthopedics, Jinshan Hospital of Fudan University, Shanghai, China
3 Department of Orthopedics, Huashan Hospital of Fudan University, Shanghai, China
|Date of Submission||09-Jun-2021|
|Date of Acceptance||09-Dec-2021|
|Date of Web Publication||20-May-2022|
No. 12, Middle Wulumuqi Road, Shanghai 200040
No. 1508, Longhang Road, Shanghai 201508
Source of Support: None, Conflict of Interest: None
Context: The growth factor receptor-bound protein 2 (Grb2)-Sos1 interaction, mediated by modular domains, plays an essential role in the oncogenic MAPK signaling pathway in osteosarcoma (OS). Recently, a dual-targeting peptide that targets the epidermal growth factor receptor and Grb2-Src homology 3 domain in OS cells was designed and synthesized.
Aims: We investigated the synergistic effects of the peptide and salinomycin (Sal), a chemotherapeutic drug with effective anti-OS properties in clinical therapy.
Subjects and Methods: Flow cytometry was used to measure the targeting efficacy of the peptide. Migration and CCK-8 assays were used to explore whether Sal and the peptide could synergistically inhibit OS cell behavior. Western blotting was used to detect apoptosis.
Statistical Analysis Used: Data were analyzed using the GraphPad Prism 5.01. Statistical analysis was performed using the Student's t-test for the direct comparisons and one-way analysis of variance for the comparisons among the multiple groups. Statistical significance was set at P < 0.05.
Results: The peptide was shown to target OS cells. When applied together, Sal and the peptide synergistically inhibited OS cell migration, invasion, and proliferation through the inhibition of Grb2-Sos1. This synergistic treatment also promoted the apoptosis of OS cells and inhibited tumor volume in vivo.
Conclusions: These data provide valuable insights into the molecular mechanisms of OS and may be beneficial in clinical therapy.
Keywords: Dual-targeting peptide, growth factor receptor-bound protein 2-Sos1 interaction, osteosarcoma, salinomycin, synergism
|How to cite this article:|
Yu Z, Xu Y, Du L, Zhang F, Shao M, Xie L, Cai G, Lyu F. Synergistic effect of growth factor receptor-bound protein 2/epidermal growth factor receptor dual-targeting peptide inhibitor and salinomycin on osteosarcoma. J Can Res Ther 2022;18:352-61
|How to cite this URL:|
Yu Z, Xu Y, Du L, Zhang F, Shao M, Xie L, Cai G, Lyu F. Synergistic effect of growth factor receptor-bound protein 2/epidermal growth factor receptor dual-targeting peptide inhibitor and salinomycin on osteosarcoma. J Can Res Ther [serial online] 2022 [cited 2022 Jun 30];18:352-61. Available from: https://www.cancerjournal.net/text.asp?2022/18/2/352/345523
| > Introduction|| |
Protein-protein interactions (PPIs) generally influence signal transduction in cancer cells through dynamic associations., These PPIs are mediated using multimodular domains, many of which bind to specific sequence motifs. An example of these interaction domains is the adaptor protein growth factor receptor-bound protein 2 (Grb2), which comprises one Src homology 2 (SH2) domain flanked by two Src homology 3 (SH3) domains.,
Son of Sevenless1 (Sos1), Ras guanine nucleotide exchange factor, and its adaptor, Grb2, are multi-domain proteins that bind fibroblast growth factor to activate the Ras-Raf/extracellular signal-regulated kinase (ERK) pathway, which is regulated by receptor tyrosine kinases (RTKs), such as epidermal growth factor receptor (EGFR)., EGFR is overexpressed in about 20% of osteosarcoma (OS) patients. It is also overexpressed on the membrane of different human malignancies and is regarded as a valuable cancer therapy target. EGFR overexpression in OS causes a poor prognosis and chemoresistance., GE11, a small peptide identified using phage display screening, was shown to have a significantly high affinity for EGFR (Kd = 22-nM), the expression of which is significantly upregulated in cancer cells. GE11 has only 12 amino acids (YHWYGYTPQNVI) and is much smaller than EGF, an EGFR ligand. Furthermore, this small peptide targets only one EGFR region. Altogether, GE11 represents a promising targeting ligand for EGFR-targeting therapy.
EGFR is activated by heterodimerization with other HER family members (HER1, HER2, HER3, and HER4). This heterodimerization causes interactions between the specific phosphotyrosine sequences of EGFR and the SH2 domains of the adaptor protein Grb2. The SH3 domain of Grb2 interacts with proline-rich sequences on targets, such as SOS, activating multiple signaling pathways, such as the Ras-Raf-MAPK pathway, which improves cellular invasion and mitogenesis.
Phosphotyrosine-containing peptide inhibitors obtained from RTK sequences inhibit the interaction between RTKs and the SH2 domain of Grb2 and reduces Raf and MAPK activation. In addition, Cussac et al. developed a proline-rich SOS-derived peptidimer inhibitor that inhibits the binding between SOS and the two SH3 domains of Grb2. Although the peptide inhibitors show some antitumor activity, the activity is transient and weak, facilitating resistance. In this work, a peptide inhibitor that targets the interaction between Sos1 (a homolog of SOS) and Grb2 by binding the SH3 domain of Grb2, guided by the high affinity between Grb2 and its native peptide ligand VPPPVPPRRR (Kd ranging 10-8 M). The typical hydrophobic cell-penetrating peptide (CPP), PFVYLI was reported to deliver peptides, liposomes, and siRNA into diverse cell lines., The CPP was coupled to the N-terminal of the Grb2 inhibitor peptide as a basic backbone. GE11 was then used to modify the backbone to allow OS cell targeting.
The chemotherapeutic drug salinomycin (Sal), isolated from Streptomyces albus, eliminates different tumors, such as liver cancer, OS, and lung cancer. Accumulating evidence has established that Sal inhibits multiple cancer stem cells through the inhibition of the Wnt/β-catenin pathway in vitro and in vivo., Therefore, Sal is regarded as a potentially effective agent for treating OS.
However, the Grb2-targeting inhibitor peptide and Sal have exhibited suboptimal therapeutic efficacy. In this study, the synergistic effect of the Grb2-targeting inhibitor peptide and Sal on OS was investigated. This dual-targeting peptide inhibits the Grb2-Sos1 interaction, resulting in the anti-proliferation of OS cells. Sal affects DNA synthesis and replication, suppressing cancer cell division. Significantly, it was discovered that the combination of Sal and the Grb2-targeting inhibitor peptide could synergistically inhibit the growth of OS cells. This synergistic approach represents a new, potentially effective treatment for OS.
| > Subjects and Methods|| |
Unless otherwise stated, all reagents were obtained from commercial enterprises and used without further purification. Rink Amide-ChemMatrix resin, Fmoc-Lys (Alloc)-OH, Fmoc-Lys (Fmoc)-OH, and other Fmoc-protected amino acids were bought from GL Biochem Ltd. (Shanghai, China), and 5 (6)-FAM (fl) was obtained from Chem-Impex International Inc. (USA). Other reagents were bought from commercial sources, including Life Technologies (USA), Sigma-Aldrich Co.(USA), and JandK Scientific Ltd.(China).
The human OS cell line U2OS, which shows high EGFR expression, was obtained from ATCC (USA). U2OS cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with high glucose, 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator.
Peptide synthesis and purification
As described previously, all peptides were synthesized using the solid-phase peptide synthesis technique based on Fmoc/HBTU chemistry. In brief, rink amide resins were swollen in DMF for 3–5 h. Then, the resins were thrilled with a 5-fold excess of amino acids, which were activated by one equivalent of 1:1 HBTU/HOBt mixture and two equivalents of DIEA in DMF. Every coupling step was sustained for 30 min at the room temperature (RT) and followed by extensive rinsing with DMF. The Fmoc group of amino acids was removed using piperidine/DMF (20% v/v) for 30 min, and the coupling/deprotection cycles were repeated.
For the Alloc group deprotection, the resins were washed with DCM five times. Next, 0.1-eq Pd (PPh3)4 and 25-eq PhSiH3 in DCM were added to the resins under N2 for 1 h. The resins were washed five times using 0.5% sodium diethyldithiocarbamate in DMF and five times using DMF. The fluorescent dye or 5 (6)-FAM (fl) was coupled to the N-terminus amino group in the presence of HBTU/HOBt. The coupling step was maintained overnight at RT. Then, the Mtt group of Fmoc-Dap (Mtt)-OH was deprotected using 1% TFA and 5% TIS in DCM, and chloroacetic acid was coupled in the presence of the activating agent HBTU/HOBt. Peptides were separated from the resin using a cleavage cocktail-containing TFA, EDT, water, and TIS (94:2.5:2.5:1, v/v) for 2 h at RT. First, ice-cold diethyl ether was used to precipitate the peptides. Next, the peptides were pelleted using centrifugation, dissolved in water, purified using semi-preparative reversed-phase HPLC, and lyophilized. Analytical reversed-phase HPLC was conducted using a Vydac (218TP54) reversed-phase HPLC column (C18, 5-μm, 4.6-mm ID × 250 mm, Alltech Associates, Inc., USA) on an LC-2010A HPLC system (Shimadzu, Kyoto, Japan).
Cellular uptake in vitro
The cellular uptake of the peptide was analyzed using flow cytometry. Briefly, U2OS cells were treated with 5 (6)-FAM (fl)-labeled peptides at a concentration of 10 μM and incubated for 2 h at 37°C. The cells were washed two to three times with ice-cold PBS. The cells were then obtained and analyzed using flow cytometry (BD Canto10C, USA) within an hour.
Cell migration assay
U2OS OS cell migration was examined using a modified Boyden chamber (6.5-mm transwells) with polycarbonate membranes (8-μm pore size) (Costar Corp., Cambridge, MA, USA). Briefly, U2OS OS cells (2.5 × 105 cells per well) were seeded overnight in a 12-well tissue culture plate and treated using PBS, GE11, TPG-GE11, Sal, or Synergy (TPG-GE11 and Sal) for 18 h. The treated cells were trypsinized, and 1 × 105 trypsinized cells suspended in DMEM were seeded in the top chambers and cultured at 37°C in 5% CO2 overnight. DMEM (600-μL) with 10% FBS was added to the lower chambers of the Transwell system. After 18 h, cells that did not migrate were eliminated from the top side of the inserts with cotton swabs. The cells that had migrated to the underside of the inserts were fixed using methanol and stained with crystal violet. Five central fields for each treatment condition were randomly selected, and cells were counted using a microscope with a 20× objective. The average number of cells in each field was calculated. Each experiment was repeated three to five times, with consistent results.
Colony formation assay
U2OS OS cells (2.5 × 105 cells per well) were seeded overnight in a 12-well tissue culture plate and treated with PBS, GE11, TPG-GE11, Sal, or Synergy (TPG-GE11 and Sal) for 48 h. The treated cells were trypsinized, and 2 × 103 cells per well were added to a six-well plate. The fresh medium was changed every 2 days. All cells were fixed with methanol and stained using crystal violet (Beyotime, Beijing, China) for 20 min after 6 days. The colonies were counted and analyzed for clonogenicity.
A total of 2.5-5 × 105 cells were added to each well of a six-well plate. An artificial wound was scratched into a monolayer of each well using a sterile plastic 200 μL micropipette. The cells were then treated using each formulation for 24 h. Next, the cells were washed thrice with sterile PBS, and serum-free medium was replaced. The medium was removed at 0 and 24 h. The widths of the scratches were observed and measured under a microscope and then photographed.
To test cell proliferation ability, the CCK-8 kit (Cell Counting Kit-8) assay was conducted according to the manufacturer's protocol. Three thousand cells per well were plated in a 96-well plate in 100-μL DMEM with 10% FBS. After 2 days, the medium was removed. CCK-8 (10-μL) and DMEM (90-μL) containing 10% FBS were mixed into each well of the 96 well plate. The plate was placed in an incubator for 4 h at 5% CO2 and 37°C. The absorbance of the wells was measured at 450 nm using a microplate reader.
U2OS cells were treated as described in the cell migration assay. First, the cells were obtained from centrifugation (2500 rpm, 5 min) and washed twice with PBS. Next, 500-μL of binding buffer and 5-μL Annexin V-FITC were added to the obtained cells. Then, 5-μL propidium iodide was added to the cell suspension at 25°C for 10 min, and the cells were analyzed using a flow cytometer within 0.5 h.
Five hundred thousand U2OS cells were seeded in a 10 cm dish. All cells were washed thrice with PBS overnight. Cells were lysed on ice in RIPA lysis buffer containing a complete protease inhibitor cocktail (Roche). The supernatants of the lysed cells were obtained using centrifugation at 12,000 × g for 10 min. Proteins were added to a 10% polyacrylamide gel for sodium dodecyl sulfatepolyacrylamide denaturing gel electrophoresis and transferred to a polyvinylidene fluoride membrane. The membranes were incubated with 5% bovine serum albumin (BSA) for 4 h at RT, 0.1% Tween 20 (TBS-T), and then for 12 h at 4°C with anti-PARP (Cat No.: 9542, Cell Signaling Technology), cleaved PARP (Cat No.: 9541, Cell Signaling Technology), Raf-1 (Cat No.: ab137435, Abcam), Phospho-Raf-1(Ser621) (Cat No.: ab157201, abcam), or GAPDH (Cat No.: ab9485, abcam) antibody diluted 1:2000 in TBS-T with 5% BSA. Membranes were washed in TBS-T for 10 min thrice and incubated for another 2 h at RT with HRP-conjugated antibody diluted at a ratio of 1:50000.
Tumor-bearing mouse model
Male BALB/c nude mice (4-6 weeks old) were bought from the Shanghai SLAC Laboratory Animal Co. Ltd. Unless otherwise noted, the mice were maintained on SPF laboratory chow under a 12 h/12 h light/dark schedule. Mice were randomly separated into five groups (five mice per group). Mice were subcutaneously injected with 5 × 106 U2OS OS cells. Intratumoral injection of a dose of PBS, 100-μM GE11, 50-μM Sal, 100-μM TPG-GE11, and Synergy (80-μM TPG-GE11 and 30-μM Sal) was administered every 3 days starting from the 15th day after cell inoculation. Xenograft tumors of mice were measured every 5 days using Vernier calipers. The tumor volume was calculated as follows: (V) = A × B2 × 0.5, where A is the minimum diameter and B is the maximum diameter. The mice were sacrificed a month later.
The Huashan Hospital of Fudan University Institutional Animal Care and Use Committee (Shanghai, China) approved this study (approval number: 2020 JS-114). All animal experiments were conducted following the protocols and guidelines of the National Institutes of Health.
Data were analyzed using GraphPad Prism 5.01 (La Jolla, CA, USA). Statistical analysis was conducted using the Student's t-test for direct comparisons and one-way analysis of variance for comparisons among multiple groups. Statistical significance was set at P < 0.05.
| > Results|| |
Design and synthesis of epidermal growth factor receptor-growth factor receptor-bound protein 2 dual-targeting peptide
A Grb2-targeting peptide with a high affinity for Grb2 of a ligand derived from the VPPPVPPRRR sequence (hSos1 1149-1158) was designed and synthesized. A CPP, PFVYLI, was added to the N-terminal of the Grb2-targeting peptide to facilitate cell entry. The EGFR-targeting peptide GE11 was used to modify the Grb2-targeting peptide to enable targeting of OS cells. Finally, TPG-GE11, Grb2, and GE11 dual-targeting peptide modified with CPP were obtained. Following cell entry, the TPG-GE11 suppressed the Grb2-Sos1 interaction, and Sal-induced DNA damage, thus, inhibiting OS cell proliferation and migration [Figure 1].
|Figure 1: The preparation of TPG-GE11 and Salinomycin (Sal) synergy system and the working mechanism of this system in OS cells. CPP: cell-penetrating peptide (PFVYLI); GE11: EGFR-targeting peptide (YHWYGYTPQNVI); Grb2-targeting peptide (VPPPVPPRRR)|
Click here to view
Cellular uptake in osteosarcoma cells
The uptake of inhibitor peptides by cells is essential for enhanced delivery efficacy. Therefore, in this study, a peptide that targets OS cells through EGFR was selected. Because of its superior stability and prolonged excitation and emission wavelengths, 5 (6)-FAM green fluorescent dye was coupled to the N-terminal amino group of the targeting peptide for cell analysis. As indicated in [Figure 2], the cellular uptake efficiency of TPG-GE11 (5-μM) and GE11 (5-μM) was determined using flow cytometry. The mean fluorescence intensity of U2OS cells treated with TPG-GE11 was 0.95 fold and 2.83 fold higher than that of cells treated with GE11 or PBS, respectively, after 2 h of incubation. Therefore, considering the improved targeting effect of GE11 coupling, TPG-GE11 was used in subsequent experiments.
|Figure 2: Targeting of U2OS OS cells by TPG-GE11. (a) Representative results of flow cytometry analysis. (b) Quantification of mean fluorescence intensity. Data are represented as the mean ± standard deviation (n = 3). Results are representative of three independent experiments. **P < 0.01|
Click here to view
Anti-proliferation and anti-invasion efficacy of synergistic treatment in osteosarcoma cells
The cytotoxic effect of the combination of TP-GE11 and Sal at final concentrations of 10-μM and 4-μM, respectively, on the proliferation and invasion of OS cells was measured. Although the proliferation assay results indicated that treatment using TP-GE11 or Sal alone weakly inhibited the proliferation of U2OS cells, the synergistic treatment dramatically suppressed proliferation at 48 h and 72 h [Figure 3]a and [Figure 3]b. The concentrations used to generate an 80% inhibition rate were as follows: TPG-GE11 (47.81376-μM) and Sal (13.10401-μM). The confidence interval was 0.5142. These results clearly illustrated the synergistic inhibition in U2OS cells [Figure 3]c and [Figure 3]d.
|Figure 3: Synergistic effects of TP-GE11 and Sal on OS cell proliferation. Synergistic inhibition of cell proliferation after 48 h (a) and 72 h (b). The effect of different concentrations of TPG-GE11 (c) or Sal (d) on the proliferation of U2OS cells. Results are representative of three independent experiments, and the error bars represent the standard deviation **P < 0.01, *P < 0.05|
Click here to view
For the scratch wound-healing assay, the extent to which the OS cells spread into the scratch area were analyzed. Treatment with TP-GE11 or Sal alone did not significantly stop the cells from closing the gap. However, synergistic treatment did significantly prevent the cells from reducing the width of the scratch, as shown by the wider distance between the two cell fronts [Figure 4]a and [Figure 4]b. These results indicated that the synergistic treatment inhibited the mobility of OS cells and thereby inhibited cell growth, presumably by inhibiting the Grb2-Sos1 interaction.
|Figure 4: Synergistic inhibition of OS cell migration. (a) Scratch wound-healing assay of U2OS cells treated using PBS, GE11, TPG-GE11, Sal, or Synergy for 24 h. (b) Quantification of the migration length of the cell front after 24 h of incubation. The black lines indicate the positions of the scratch and the cell front at time zero. Results are representative of three independent experiments, and the error bars represent the standard deviation **P < 0.01, *P < 0.05|
Click here to view
Anti-migratory and anti-clonogenic effect of synergistic treatment on osteosarcoma cells
A migration assay was conducted to determine whether treatment with TP-GE11 and Sal can suppress Grb2-Sos1signaling. This inhibition may redirect the downstream Raf/MAPK signaling and suppress the migration of OS cells with high EGFR expression. Different groups of U2OS cells were seeded in the upper chamber of a Transwell apparatus, and cells migrating to the lower side of the filter were counted.
Although treatment with TPG-GE11 (10-μM) or Sal (4-μM) alone inhibited the migration of U2OS cells, the inhibition rate was not significant. However, the synergistic treatment greatly reduced the migratory behavior of OS cells [Figure 5]a and [Figure 5]b. This showed that TPG-GE11 combined with Sal exerted a synergistic inhibitory effect on the migration of OS cells. Consistently, the colony formation assay, as shown in [Figure 5]c and [Figure 5]d, indicated that the proliferation rate of synergistically treated cells decreased relative to that of cells treated with TPG-GE11, Sal, or PBS. Western blotting was conducted to quantify the level of the Raf-1 and phospho-Raf-1 in OS cells. The combination of TPG-GE11 and Sal was the most effective treatment, resulting in the low expression of phospho-Raf-1 [Supporting Information Figure S1]. These results showed that the TPG-GE11 peptide modulator redirected the downstream signaling pathway of Grb2-Sos1 in OS. The combination of the TPG-GE11 peptide and Sal may be a novel strategy for OS treatment.
|Figure 5: (a) The number of stained cells that migrated to the bottom side of the membrane of the Transwell chamber, counted under a microscope. The number of migrated cells significantly reduced with the TP-GE11 and Sal-combined treatment. (b) Quantification of cancer cells receiving different treatments. (c) Colony formation assay was conducted with various treatments. (d) Statistical analysis of colony formation assay results (c). Results are representative of three independent experiments, and the error bars represent the standard deviation **P < 0.01, *P < 0.05. Scale bar: 100-μm|
Click here to view
Apoptotic effect of synergistic treatment on osteosarcoma cells
To determine the apoptotic effect of the synergistic treatment, an Annexin V-FITC assay was conducted. Flow cytometry results indicated a significant increase in apoptosis in the synergistic treatment group than that in the PBS group. It was found that TPG-GE11 treatment and Sal treatment caused apoptotic rates of about 16% and 25%, respectively, when separately administered, while the apoptotic rate induced by synergistic treatment increased to about 40% [Figure 6]a and [Figure 6]b.
|Figure 6: Synergistic effects of TPG-GE11 and Sal on apoptosis in U2SO OS cells.(a) Annexin V/PI flow cytometry of U2SO cells treated separately or concurrently with TPG-GE11 and Sal.(b) Quantification of the apoptosis rate in selected cells.(c) Western blotting analysis showing the expression levels of cleaved PARP in U2SO cells.(d) Quantification of the apoptosis rate in U2SO cells. Results are representative of three independent experiments, and the error bars represent the standard deviation. **P < 0.01, *P < 0.05|
Click here to view
Western blotting was then conducted to quantify the level of the cell-specific apoptosis marker (cleaved PARP) in OS cells. Significantly, the mixture of TPG-GE11 and Sal was the most effective treatment, leading to a high expression of cleaved PARP [Figure 6]c and [Figure 6]d. This indicates a synergistic effect. In contrast, treatment with TPG-GE11 (10-μM) or Sal (4-μM) alone led to much lower expression of cleaved PARP than that in the synergistic treatment group. In conclusion, these data show that synergistic treatment enhanced Grb2 expression, inducing OS cell apoptosis.
Suppressive effect of synergistic treatment on osteosarcoma in vivo
To investigate the effect of synergistic treatment in vivo, U2SO cells were subcutaneously injected into BALB/c nude mice. After tumors attained approximately 100 mm3 size, mice were randomly selected to receive treatment with PBS, GE11, TPG-GE11, Sal, or Synergy for 3 days in 21 days. Tumor volume was detected every 5 days for up to 30 d [Figure 7]a. It was found that TPG-GE11 or Sal treatment suppressed tumor development compared to that in the PBS or GE11 groups. With synergistic treatment, tumor growth suppression was more significant, with a substantial reduction in tumor volume and weight than in the other groups [Figure 7]b and [Figure 7]c.
|Figure 7: The suppressive effect of synergistic treatment on tumor growth in nude mice. (a) Representative image of the dissected xenograft tumors of the mice, treated as shown. (b) Time-dependent tumor growth in nude mice. (c) Statistical analysis of the tumor weight of each group. Results are representative of three independent experiments, and the error bars represent the standard deviation **P < 0.01, *P < 0.05|
Click here to view
| > Discussion|| |
OS is the most common type of invasive and malignant bone sarcoma in children and adolescents., More than 85% of malignant OS occur in the lungs, while others occur in distant tubular long bones. Metastatic OS has a poor prognosis, with a low 5-year overall survival rate, owing to chemoresistance and complications, such as lung calcification. Although treatments for OS have developed over the past two decades, including, surgery, chemotherapy, and adjuvant chemotherapy, no meaningful improvement has been made. Because of the strong specific killing effect and fewer adverse effects, targeted therapy has been used as a treatment for OS. Geller et al. developed a novel OS therapy using radioimmunotherapy that targets insulin-like growth factor-2 receptor, which is ubiquitously expressed in OS. Tian et al. developed CD271 antibody-functionalized PEGylated multifunctional hollow gold nanospheres (HGNs) for targeted therapy of OS. Targeted treatment using HGNs attains excellent cell viability inhibition compared with that in nontargeted treatment groups.
The adaptor protein Grb2 is highly expressed in several cancers, including breast cancer, OS, and liver cancer. The N-terminal SH3 domain of Grb2 is constitutively associated with the guanine nucleotide exchange factor Sos1, which then links to RTKs and activates the RAS and ERK/MAPK downstream signaling pathways, causing cancer cell proliferation, differentiation, migration, and invasion., Therefore, Grb2 is considered an ideal target for treating OS.
EGFR is overexpressed in patients with OS and is regarded as a valuable OS cancer therapy target. Several antibodies and small molecules have been demonstrated to target EGFR and exhibit superior therapeutic efficacy in various cancers, including liver cancer, breast cancer, and OS., The small peptide GE11 (YHWYGYTPQNVI), identified by phage display screening, can effectively target EGFR in OS. GE11 was used to promote inhibitor delivery to EGFR-overexpressing OS cells.
It has been more than 30 years since the discovery of CPPs. CPPs have been widely applied for delivering numerous therapeutic reagents, including peptides, siRNA, and proteins, in vitro and in vivo. A common hydrophobic penetration peptide (PFVYLI) exhibited excellent performance in delivering siRNA and peptides into various cell lines., In this study, a Grb2 (VPPPVPPRRR) peptide inhibitor coupled with a CPP (PFVYLI) was synthesized as the basic backbone. GE11 was then used to modify the backbone to allow EGFR targeting in OS cells.
The presence of GE11 is vital for maintaining the targeting efficacy of TPG-GE11 in OS cells highly expressing EGFR. As demonstrated by FACS, TPG-GE11 could efficiently bind to OS cells and improve the cytotoxic effect compared to that induced by PBS. Our proliferation, invasion, and migration analysis demonstrated that TPG-GE11 possessed significant cytotoxic effects on OS compared with PBS and GE11. Although the peptide inhibitors showed specific antitumor activity when administered alone, their action was transient and weak.
Sal has been used as an effective chemotherapeutic agent against numerous cancers, including breast cancer, prostate cancer, and liver cancer.,, Moreover, Sal inhibits the proportion of breast cancer stem cells by >100 fold compared to the chemotherapeutic drug paclitaxel. Importantly, Sal has been used to achieve partial regression of cancer metastasis with few side effects. Therefore, Sal represents a promising chemotherapeutic drug against OS. Our proliferation, invasion, and migration analysis demonstrated that Sal exerted significant cytotoxic effects on OS, compared with PBS, GE11, and TPG-GE11. In this study, the synergistic effect of the Grb2-targeting inhibitor peptide and Sal on OS was exploited. This dual-targeting peptide inhibited the progression of OS in vitro and in vivo.
| > Conclusion|| |
The novel conjugated peptide TPG-GE11 selectively suppressed Grb2 by targeting EGFR in OS cells. The results confirmed that the TPG-GE11 peptide enhanced drug efficacy. Consistently, the synergistic effect of TPG-GE11 and Sal on Grb2 during OS development in vitro and in vivo was observed. These findings provide valuable insights into the molecular mechanisms of Grb2. Synergistic treatment could serve as an effective therapy for OS.
Financial support and sponsorship
This work was financially supported by the National Natural Science Foundation of China (81602358).
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Pawson T, Nash P. Assembly of cell regulatory systems through protein interaction domains. Science 2003;300:445-52.
Scott JD, Pawson T. Cell signaling in space and time: Where proteins come together and when they're apart. Science 2009;326:1220-4.
McDonald CB, Seldeen KL, Deegan BJ, Lewis MS, Farooq A. Grb2 adaptor undergoes conformational change upon dimerization. Arch Biochem Biophys 2008;475:25-35.
Yu Y, Nie Y, Feng Q, Qu J, Wang R, Bian L, et al
. Targeted covalent inhibition of Grb2-Sos1 interaction through proximity-induced conjugation in breast cancer cells. Mol Pharm 2017;14:1548-57.
Buday L, Downward J. Epidermal growth factor regulates p21ras through the formation of a complex of receptor, Grb2 adapter protein, and Sos nucleotide exchange factor. Cell 1993;73:611-20.
Haines E, Saucier C, Claing A. The adaptor proteins p66Shc and Grb2 regulate the activation of the GTPases ARF1 and ARF6 in invasive breast cancer cells. J Biol Chem 2014;289:5687-703.
Lee JA, Ko Y, Kim DH, Lim JS, Kong CB, Cho WH, et al
. Epidermal growth factor receptor: Is it a feasible target for the treatment of osteosarcoma? Cancer Res Treat 2012;44:202-9.
Pahl JH, Ruslan SE, Buddingh EP, Santos SJ, Szuhai K, Serra M, et al
. Anti-EGFR antibody cetuximab enhances the cytolytic activity of natural killer cells toward osteosarcoma. Clin Cancer Res 2012;18:432-41.
Genta I, Chiesa E, Colzani B, Modena T, Conti B, Dorati R. GE11 peptide as an active targeting agent in antitumor therapy: A minireview. Pharmaceutics 2017;10:2.
Hu D, Mezghrani O, Zhang L, Chen Y, Ke X, Ci T. GE11 peptide modified and reduction-responsive hyaluronic acid-based nanoparticles induced higher efficacy of doxorubicin for breast carcinoma therapy. Int J Nanomedicine 2016;11:5125-47.
Brandt BH, Roetger A, Dittmar T, Nikolai G, Seeling M, Merschjann A, et al
. c-erbB-2/EGFR as dominant heterodimerization partners determine a motogenic phenotype in human breast cancer cells. FASEB J 1999;13:1939-49.
Kozer N, Barua D, Henderson C, Nice EC, Burgess AW, Hlavacek WS, et al
. Recruitment of the adaptor protein Grb2 to EGFR tetramers. Biochemistry 2014;53:2594-604.
Yamazaki T, Zaal K, Hailey D, Presley J, Lippincott-Schwartz J, Samelson LE. Role of Grb2 in EGF-stimulated EGFR internalization. J Cell Sci 2002;115:1791-802.
Chardin P, Camonis JH, Gale NW, van Aelst L, Schlessinger J, Wigler MH, et al
. Human Sos1: A guanine nucleotide exchange factor for Ras that binds to GRB2. Science 1993;260:1338-43.
Cussac D, Vidal M, Leprince C, Liu WQ, Cornille F, Tiraboschi G, et al
. A Sos-derived peptidimer blocks the Ras signaling pathway by binding both Grb2 SH3 domains and displays antiproliferative activity. FASEB J 1999;13:31-8.
Cai D, Gao W, He B, Dai W, Zhang H, Wang X, et al
. Hydrophobic penetrating peptide PFVYLI-modified stealth liposomes for doxorubicin delivery in breast cancer therapy. Biomaterials 2014;35:2283-94.
Park JW, Bang EK, Jeon EM, Kim BH. Complexation and conjugation approaches to evaluate siRNA delivery using cationic, hydrophobic and amphiphilic peptides. Org Biomol Chem 2012;10:96-102.
Antoszczak M. A comprehensive review of salinomycin derivatives as potent anticancer and anti-CSCs agents. Eur J Med Chem 2019;166:48-64.
Klose J, Eissele J, Volz C, Schmitt S, Ritter A, Ying S, et al
. Salinomycin inhibits metastatic colorectal cancer growth and interferes with Wnt/β-catenin signaling in CD133+
human colorectal cancer cells. BMC Cancer 2016;16:896.
Wickström M, Dyberg C, Milosevic J, Einvik C, Calero R, Sveinbjörnsson B, et al
. Wnt/β-catenin pathway regulates MGMT gene expression in cancer and inhibition of Wnt signalling prevents chemoresistance. Nat Commun 2015;6:8904.
Markowska A, Kaysiewicz J, Markowska J, Huczyński A. Doxycycline, salinomycin, monensin and ivermectin repositioned as cancer drugs. Bioorg Med Chem Lett 2019;29:1549-54.
Ma W, Sha SN, Chen PL, Yu M, Chen JJ, Huang CB, et al
. A cell membrane-targeting self-delivery chimeric peptide for enhanced photodynamic therapy and in situ
therapeutic feedback. Adv Healthc Mater 2020;9:e1901100.
Wang W, Zhao HF, Yao TF, Gong H. Advanced development of ErbB family-targeted therapies in osteosarcoma treatment. Invest New Drugs 2019;37:175-83.
Yang M. Prognostic role of pathologic fracture in osteosarcoma: Evidence based on 1,677 subjects. J Cancer Res Ther 2015;11:264-7.
Bacci G, Briccoli A, Rocca M, Ferrari S, Donati D, Longhi A, et al
. Neoadjuvant chemotherapy for osteosarcoma of the extremities with metastases at presentation: Recent experience at the Rizzoli Institute in 57 patients treated with cisplatin, doxorubicin, and a high dose of methotrexate and ifosfamide. Ann Oncol 2003;14:1126-34.
Geller DS, Morris J, Revskaya E, Kahn M, Zhang W, Piperdi S, et al
. Targeted therapy of osteosarcoma with radiolabeled monoclonal antibody to an insulin-like growth factor-2 receptor (IGF2R). Nucl Med Biol 2016;43:812-7.
Tian J, Gu Y, Li Y, Liu T. CD271 antibody-functionalized HGNs for targeted photothermal therapy of osteosarcoma stem cells. Nanotechnology 2020;31:305707.
Verbeek BS, Adriaansen-Slot SS, Rijksen G, Vroom TM. Grb2 overexpression in nuclei and cytoplasm of human breast cells: A histochemical and biochemical study of normal and neoplastic mammary tissue specimens. J Pathol 1997;183:195-203.
Joffre C, Barrow R, Ménard L, Calleja V, Hart IR, Kermorgant S. A direct role for Met endocytosis in tumorigenesis. Nat Cell Biol 2011;13:827-37.
Nicholson RI, Gee JM, Harper ME. EGFR and cancer prognosis. Eur J Cancer 2001;37 Suppl 4:S9-15.
Oba M. Cell-penetrating peptide foldamers: Drug-delivery tools. Chembiochem 2019;20:2041-5.
Mai TT, Hamaï A, Hienzsch A, Cañeque T, Müller S, Wicinski J, et al
. Salinomycin kills cancer stem cells by sequestering iron in lysosomes. Nat Chem 2017;9:1025-33.
Pan Y, Ma S, Cao K, Zhou S, Zhao A, Li M, et al
. Therapeutic approaches targeting cancer stem cells. J Cancer Res Ther 2018;14:1469-75.
Sajwani FH. Frondoside A is a potential anticancer agent from sea cucumbers. J Cancer Res Ther 2019;15:953-60.
Zhang Y, Zhang H, Wang X, Wang J, Zhang X, Zhang Q. The eradication of breast cancer and cancer stem cells using octreotide modified paclitaxel active targeting micelles and salinomycin passive targeting micelles. Biomaterials 2012;33:679-91.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]