A synthetic peptide inhibits human ovarian cancer cell motility

Abstract

Phage display libraries have been commonly used to identify recognition sites and antigen–antibody binding systems in tumor cells since the 1990s. In the previous study, we isolated a 12-mer binding peptide SVSVGMKPSPRP (SV-peptide) while isolating human ovarian cancer cells (SK-OV-3) using a random phage display library. Therefore, we tested the specific binding and anticancer properties of the SV-peptide with respect to cell viability and motility. The peptide-binding assay showed the immunofluorescent staining intensity of tumor cells (A2780, A549 and SK-OV-3) > control cells (HUVEC). Monolayer scratch healing and Transwell transmigration assays showed that the SV-peptide inhibited the migration of all three cancer cell types but not HUVEC. The mechanism involves a suppression of cytoskeletal F-actin filament arrangement into filopodia and lamellipodia through downregulation of Rac1 but not RhoA. The FAK–PI3K signaling pathway was also affected by the peptide. Western blot analysis revealed a downregulation of FAK, but not of PI3K, after 24 h incubation of SK-OV-3 cells with the SV-peptide. Metalloproteinase-2 (MMP-2) was also downregulated by the SV-peptide. Finally, the viability of cancer cell lines (SK-OV-3 and A549) decreased in the presence of the SV-peptide after 48 h. Transmission electron microscopy of the SV-peptide-exposed cancer cultures showed extensive vacuolization, swollen mitochondria, and rough endoplasmic reticulum. Thus, the SV-peptide has some certain application prospects in the tumor therapy.

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1. Introduction

The development of the phage display library technology has led to the discovery of various small biomolecules (peptides), serving as potential reagents, for diverse targets of interest.^1–7 These biomolecules possess excellent properties, such as high affinity, strong specificity for their targets, better cell penetration, and low toxicity.^8–12 The anticancer peptides identified with this technology include necrotic, apoptotic, function-blocking, antiangiogenic, and immunostimulatory peptides.

Among these, the 12-mer binding peptide SVSVGMKPSPRP (SV-peptide) raised much attention and controversy because it bound to various targets from distinct fields, including inorganic materials (hydroxyapatite),¹³ Fe–Pt alloy biosensing nanoparticles,¹⁴ semiconductors (GaAs–InP),¹⁵ tumor cells (human lung and oral cancer cells),¹⁶ DNA,¹⁷ human monoclonal immunoglobulin M (IgM),¹⁸ and HIV-1 TAR RNA element.¹⁹ Therefore, it is believed that the SV-peptide has no specificity and would be of little value, and the amplification rate of the phage clones displaying the SV-peptide was faster than other phage clones.²⁰ However, the SV-peptide was recently found to bind tumor neovasculature, rather than to normal blood vessels, in severe combined immunodeficiency mice bearing human tumors.¹⁶ In addition, the SV-peptide bound specifically to non-small cell lung cancer (NSCLC) cell lines and surgical specimens from lung cancer patients.²¹ Therefore, this biomolecule may be more promising for cancer cell targeting than expected.

In a recent study, the SV-peptide (named peptide ZP1) was identified as an ovarian tumor-specific peptide by biopanning on the SK-OV-3 ovarian carcinoma cell line with a phage display peptide library.²² The high specificity of the SV-peptide for SK-OV-3 cell line suggests that it can be used as a biomarker for targeting drug delivery and as a drug for tumor therapy. Therefore, the aim of the present study was to evaluate the effects of the SV-peptide on the migration and viability of tumor and control cells.

2. Results and discussion

2.1 Specificity of synthetic peptide binding to tumor cells

The ability of the SV-peptide to interact with human cells in vitro was determined by peptide-binding assay using biotin–SV-peptide and biotin–ARP-peptide (specificity control), A2780, A549, and SK-OV-3 cancer cell lines, and HUVEC. Immunofluorescent staining showed that the biotin–SV-peptide binds to the surface of SK-OV-3, A549, and HUVEC cells but is internalized by A2780 cells (Fig. 1A–C). Fluorescence intensity was ranked in the order of tumor cells (A2780, A549 and SK-OV-3) > HUVEC (Fig. 1D). In contrast, the ARP-peptide did not bind to any cell lines (data not shown). These data suggest that the SV-peptide preferred to bind to tumor cells than control cells. Specific bindings are one of the superiorities of the peptide isolated from the targets of interest using a phage display library. Likewise, the SV-peptide was previously reported to bind SK-OV-3 cells but with higher affinity than other ovarian tumor cell line A2780, human osteosarcoma (MG63) cells, and non-cancer cell lines.²² The apparent discrepancy in the relative affinities to the ovarian cancer cell lines (SK-OV-3 and A2780) may be related to assay conditions that could detach more loosely bound abundant peptides. Nonetheless, both studies confirm the affinity of the SV-peptide for ovarian cancer cells.

Fig. 1 Images of biotin–SV-peptide binding to control and cancer cell lines using immunofluorescence staining. (A and e) SK-OV-3, (B and f) A2780, (C and g) A549, and (D and h) HUVEC cells were incubated with biotin–SV-peptide, followed by avidin–FITC. The nuclei were stained with Hoechst 33258 (blue; e–h). The cells were observed with a laser scanning confocal microscope (Leica, Germany). The magnification bar represents 25 μm.

The fact that the SV-peptide interacted with all three cancer cell lines suggests that it recognizes common features related to tumorigenesis. The high binding affinity of the SV-peptide to tumor cells was ascribed to its proline–serine–proline (PSP) motif.¹⁶ While the PSP motif possesses homology with DNA binding motifs and predicted protein interaction and in human proteins, it was not identified as a known ligand for membrane proteins by Blast search.¹⁶ Therefore, the mechanism SV-peptide–cancer cell interaction remains unidentified.

2.2 Effect of SV-peptide on cancer cell migration

The effect of the SV-peptide on cancer cell migration was determined using a monolayer scratch wound migration assay. Fig. 2A shows that tumor cell migration (SK-OV-3, A2780, and A549) was significantly inhibited after 24 h incubation with the SV-peptide. Quantitative analysis indicated that the migration distance of SK-OV-3 cells was significantly shorter than for control HUVEC cells and other tumor cells after 4 h incubation (Fig. 2B, p < 0.05). Furthermore, the migration distance of A2780 cells was the shortest of all three tumor cell lines at 24 h (p < 0.05). These data demonstrate that the SV-peptide could inhibit the migration of ovarian cancer cells from different origins, including ascites (SK-OV-3) or tumors (A2780). In contrast, the migration distance of HUVEC was significantly longer in the presence of the SV-peptide at 4 h and 24 h (p < 0.05, Fig. 2C). Altogether, these data demonstrate that the SV-peptide could inhibit tumor cell migration but promote HUVEC migration.

Fig. 2 Effects of SV-peptide on the migration of control and cancer cell lines by the scratch wound healing assay. (A) Images showing cell migration at 0, 4, and 24 h (magnification ×40). (B) Migration distance at 4 and 24 h. (C) Average amount of migrated cells across the baseline [dashed line in (A)]. The white and black histograms indicate the amount of migrated untreated and treated cells, respectively. Asterisk denotes statistically significant difference with p < 0.05.

Cell migration was also investigated with the Transwell assay. Fig. 3 shows that the SV-peptide significantly reduced the number of SK-OV-3, A2780, and A549 cells that migrated across the transwell insert membrane at 24 h (p < 0.05). While the peptide did not affect the migration rate of HUVEC, the cells tended to bind to each other and form mesh structures. These data suggested that the SV-peptide may play an active role in the in vitro vascularization process of HUVEC. In contrast, cell migration was not influenced by the ARP-peptide (data not shown).

Fig. 3 Effects of SV-peptide on the migration of control and cancer cell lines using Transwell assay. (A and e) SK-OV-3, (B and f) A2780, (C and g) A549 and (D and h) HUVEC cells. (e–h) Represented the cells treated with 0.1 mg mL⁻¹ SV-peptide and (A–D) represented the untreated cells. The cells that migrated across the membrane (8 μm pores) were stained purple with crystal violet and photographed with an inverted phase contrast microscope (magnification, ×200). (I) The OD₅₇₀ of the different treated cells and the control group, respectively. Asterisk denotes statistically significant difference with p < 0.05.

The cytoskeleton plays a very important role in cell migration,^23,24 which is orchestrated by numerous integrins, F-actin, and matrix metalloproteinases (MMPs).^25–27 Therefore, we tested whether F-actin filaments of the cytoskeleton are involved in the SV-peptide-mediated inhibition of cancer cell migration. Immunofluorescence staining revealed abundant F-actin containing filopodia and lamellipodia structures in SK-OV-3 cells (Fig. 4A). The SV-peptide significantly reduced filopodia and lamellipodia staining after 24 h incubation (Fig. 4B). Moreover, filamentous fibers in the cytoskeleton depolymerized. However, there were abundant F-actin containing filopodia and lamellipodia structures in HUVEC cells when treated with the SV-peptide for 24 h (Fig. 4D, white arrows). In Fig. S4,† the SV-peptide significantly reduced filopodia and lamellipodia staining after 24 h incubation with A549 and A2780 cell lines. These data indicated that the SV-peptide inhibited cell migration through changes in the cytoskeletal structure of SK-OV-3, A549 and A2780 cells. On the contrary, SV-peptide promoted the HUVEC cell migration through stimulating the expression of F-actin.

Fig. 4 Immunofluorescence analyses of the effects of SV-peptide on the expression and distribution of F-actin. (A) SK-OV-3 cells, (B) SK-OV-3 cells were treated with SV-peptide for 24 h. (C) HUVEC cells, (D) HUVEC cells were treated with SV-peptide for 24 h. F-actin was stained with Alexa Fluor 488 phalloidin. The magnification bar represents 25 μm.

During cell migration, cytoskeletal changes are regulated primarily by small G proteins, namely Rac1 for lamellipodia, RhoA for stress fibers, and Cdc42 for filopodia.²⁸ Cdc42 and Rac1 induce cell polarization and lamellipodium formation at the leading edge, respectively, whereas RhoA facilitates cell contraction. Western blot analysis was conducted to test the effects of the SV-peptide on Rac1 and RhoA expression in SK-OV-3 cells (Fig. 5A). There was no significant difference in RhoA protein expression between control and treated cells. However, Rac1 was downregulated after the cells were treated for 24 h with the SV-peptide. These data are consistent with the F-actin immunofluorescence staining. More importantly, they revealed that the SV-peptide specifically targets small G proteins, promoting cell migration.

Fig. 5 Effects of SV-peptide on the expression and distribution of small G proteins (Rac1 and RhoA) in SKOV3 cells. (A) Western blot analysis of Rac1, RhoA, FAK, and PI3K protein expression. (B) Immunofluorescence analysis of Rac1 protein expression. (C) Immunofluorescence analysis of RhoA protein expression. The nucleus was stained with Hoechst 33258 (blue). The magnification bar represents 25 μm.

Immunofluorescence staining showed that Rac1 was concentrated in the cytoplasm, while RhoA was distributed in the cytoplasm and nucleus (Fig. 5B and C). The SV-peptide did not affect Rac1 distribution but RhoA moved to the edges in areas of lamellipodia formation. Staining intensity analysis was consistent with the western blots. The SV-peptide did not affect RhoA but caused a significant decrease in Rac1 immunofluorescence.

Cyclic peptides containing the sequence HWGF could specifically bind to MMP-2 and MMP-9 rather than to other MMP family members. In addition, the synthetic peptide CTTHWGFTLC inhibited the migration of tumor and human endothelial cells.²⁹ In the study, the expression of F-actin, Rac1, and RhoA of the SK-OV-3 cells reduced after incubation with the SV-peptide for 24 h. The FAK signaling pathway regulates cell migration through integrin-mediated signal transduction.^30–33 When PI3K binds to the autophosphorylation site of FAK at Y397, the PI3K–AKT complex initiates signals regulating cell viability and migration.^32,34,35 In the present study, western blot analysis indicated that the SV-peptide downregulated FAK but not PI3K (Fig. 5A). These data suggest that the SV-peptide inhibits cancer cell migration by interfering with formation of the PI3K–AKT complex in the FAK–Rho GTPases signaling pathway.

Human MPPs participate in various cell functions, including invasion, migration and angiogenesis. Among them, MMP-2 was found particularly important for cancer cell migration. Therefore, we tested the effect of the SV-peptide on MMP expression in SK-OV-3 cells with ELISA. Fig. 6A shows that the SV-peptide downregulated MMP-2 in a dose-dependent manner during the first 48 h, starting at 0.02 mg mL⁻¹. After 48 h, MMP-2 expression started to increase in all cultures but at a lower rate in SV-peptide-treated cultures than control cultures. The SV-peptide also downregulated MMP-9 in a dose-dependent manner over 24 h (Fig. 6B). Altogether, these experiments suggest that SV-peptide-mediated inhibition of cancer cell migration involves the inhibition of FAK–Rho signaling and the downregulation MMP-2 and MMP-9 expression.

Fig. 6 Effects of SV-peptide on the expression of (A) MMP-2 and (B) MMP-9. SK-OV-3 cells were treated with SV-peptides for 24 h and 48 h and their expression was measured using enzyme-linked immunosorbent assay (ELISA).

2.3 Effect of SV-peptide on cancer cell viability

The MTT assay was used to compare the effect of the SV-peptide on the viability of control cells (HUVEC) and cancer (SK-OV-3, A549, and MG63) cell lines. Fig. S1† shows that the viability of cancer cells (SK-OV-3 and A549) decreased in the presence of the SV-peptide after 48 h and 72 h (p < 0.05). However, the viability of SK-OV-3 cells was only reduced by the highest SV-peptide concentration (0.5 mg mL⁻¹) at 72 h. In contrast, A549 cells were significantly affected starting at a very low concentration of the SV-peptide (0.0025 mg mL⁻¹), and the effects increased with peptide concentration (p < 0.05). Therefore, A549 cells were more sensitive to SV-peptide than SK-OV-3 cells. It is noteworthy that the viability of HUVEC and MG63 cells increased starting at very low SV-peptide concentrations SV-peptide (0.001 mg mL⁻¹; p < 0.05). In contrast, the ARP peptide either increased cell viability (p < 0.05) or had no effect (Fig. S2†). It was suggested that peptides, isolated from tumor cells with a phage display library, reduce the viability of tumor cells by damaging structures or via indirect mechanisms (i.e., pore formation), leading to cell death.^36,37

The ultrastructure of control and cancer cells exposed to the SV-peptide was examined with TEM. After 72 h exposures to 0.1 mg mL⁻¹ SV-peptide, SK-OV-3 (Fig. S3B†), A2780 (Fig. S3D†), and A549 (Fig. S3F†) cells contained swollen mitochondria with distended rough endoplasmic reticulum (RER). In addition, the plasma membrane of all tumor cell lines was ruptured and showed extensive vacuolization. Such characteristics were typical of apoptosis or cell death. In contrast, HUVEC presented a normal organization with higher numbers of mitochondria (Fig. S3H†). These data suggest that the SV-peptide promotes the growth of HUVEC. These results were consistent with previous studies.³⁸

3. Experimental

3.1 Cells and regents

In the study, ovarian carcinoma cell lines (SK-OV-3 and A2780), the lung adenocarcinoma cell line (A549), the human osteosarcoma cell lines (MG63) and human umbilical vein cell line (HUVEC) were used. SK-OV-3 and A2780 were purchased from West China Second University Hospital, China. A549 was gifted by the Key Laboratory of Bio-resources and Eco-environment, the Ministry of Education, the School of Life Sciences, Sichuan University, China. MG63 and HUVEC were obtained from the Institute of Biomedical Engineering, Sichuan University, China. All cells were cultured in Dulbecco's modified Eagle medium (DMEM, Gibco, USA) with 15% fetal calf serum (FCS, Hyclone, USA) and maintained in a humidified incubator (5% CO₂, 37 °C).

The peptides SVSVGMKPSPRP (SV-peptide), biotin-labeled SVSVGMKPSPRP (biotin–SV-peptide), ARPLEHGSDKAT (ARP-peptide), and biotin-labeled ARPLEHGSDKAT (biotin–ARP-peptide) were synthesized by the Shanghai Biotech Bioscience and Technology Co., Ltd (Shanghai, China). Peptide purity was confirmed to be >95% using high pressure liquid chromatography (HPLC, SHIMADZU, Japan) based on their molecular mass.

The primary antibodies were anti-PI3K mouse monoclonal antibody (mAb, sc-100407), anti-FAK (A-17) rabbit mAb (sc-1688), anti-Rac1 (C-11) rabbit mAb (sc-59), and anti-RhoA (26C4) mouse mAb (sc-418). They were purchased from Santa Curz™ Biotechnology, Inc. (CA, USA). The peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG (secondary antibody) were purchased from Dingguo Biotechnology Co., Ltd (Beijing, China). The secondary fluorescence isothiocyanate (FTIC)-conjugated goat anti-mouse IgG was purchased from Biosynthesis Biotechnology Co., Ltd (Beijing, China), and Alexa Fluor 488 phalloidin (A12379) was purchased from Invitrogen Life Technologies Corporation (USA).

Enzyme-linked immunosorbent assay (ELISA) kits for human matrix MMP-2 (DMP2F0) and MMP-9 (DMP900) were purchased from R&D Systems, Inc. (USA).

3.2 Identification of synthetic peptides binding to cancer cells using the immunofluorescence assay

This protocol was performed as described previously.¹⁹ In brief, SK-OV-3, A2780, A549, and HUVEC cells in the logarithmic phase were seeded and grown at 80% confluence on the chamber slides. They were cultured with serum-free medium (2 h) and then fixed with 3% paraformaldehyde (10 min). The biotin–SV-peptide and biotin–ARP-peptide mixture (100 μL; 0.1 mg mL⁻¹) was added to each chamber slide and incubated with the cells for 30 min. After washing with phosphate-buffered saline (PBS, pH 7.2), the peptides were stained for 30 min with a 1 : 60 dilution of Avidin–FITC (Wuhan Boster Bio-Engineering Co., China). The cells were counterstained with Hoechst 33258 (Sigma, USA), mounted with PBS, and observed with a laser scanning confocal microscope (Leica TCS SP5, Germany).

3.3 Cells migration assay–scratch wound healing assay

The effect of the SV-peptide on cell motility was studied using a monolayer scratch wound healing assay with SK-OV-3, A2780, A549, and HUVEC, respectively.³⁹ In brief, 400 μL fibronectin (40 μg mL⁻¹) was added to each well of a 12-well plate (BD Biosciences, USA) and incubated overnight (4 °C). Then 1 mL serum-free culture medium containing 0.1% bovine serum albumin (BSA, Beyotime Institute of Biotechnology, China) was added, and the plate was incubated (37 °C, 1 h) to dehydrate the membrane. Each well received 500 μL (1 × 10⁵) of cells, which were serum-starved for 12 h after they reached 90% confluence. During the last 4 h, hydroxyurea was added to block DNA synthesis at a final concentration of 6 mmol L⁻¹. After the 12 h incubation, uniform scratches were made in the cell monolayer with a micropipette tip (Corning, USA). The monolayers were washed gently with PBS and photographed with an inverted phase contrast microscope (time = 0 min, Olympus, Japan). Then, 2 mL serum-free culture medium containing SV-peptide solution (0.1 mg mL⁻¹) was added to the treated group and 2 mL serum-free culture medium containing PBS solution (0.1 mg mL⁻¹) was added to the control group. Images of wound closing were acquired after 4 h and 24 h incubations under static conditions (5% CO₂, 37 °C) without serum. Width of each scratch was measured at six randomly chosen locations at the three time-points (0, 4, and 24 h). The rate of cell migration was calculated from the difference in width at 4 h and 24 h relative to the width at 0 h. Each measurement was conducted in triplicate in each field.

Cell migration was also assessed with SK-OV-3, A2780, A549, or HUVEC using 24-well transwell inserts (8 μm pore size polycarbonate membrane; Corning, USA).⁴⁰ In brief, cells were washed with PBS, resuspended in serum-free medium, deposited in each upper chamber (1 × 10⁵ cells), and allowed to migrate through the insert membrane toward the lower chamber. The lower chamber was filled with 0.6 mL DMEM medium containing 20% serum and 15% SV-peptide or ARP-peptide as a chemoattractant. During the migration, the transwells were incubated for 24 h at 37 °C. The membranes were washed with PBS, and the cells remaining on the upper surface were wiped away with cotton swabs. The cells that migrated to the lower surface of the membranes was stained with 0.1% crystal violet (Sigma, USA) solution, and photographed with an inverted phase contrast microscope (Olympus, Japan, 200×). Further, the membranes were mounted in glacial acetic acid (1 : 2 in PBS) and the absorbance of the crystal violet was measured at 490 nm with a microplate reader (Molecular Devices, USA).

3.4 Western blot and immunofluorescence analyses

SK-OV-3 cells were seeded (10 000 cells per mm²) on a Petri dish (60 mm) for 24 h, then 400 μL SV-peptide (0.1 mg mL⁻¹) solution was added to the treated group (peptide and cancer cells), and 400 μL PBS to the control group (only cancer cells). After 24 h of culture, the cells were washed three times with PBS and dissociated with 50 μL cell lysis solution containing 1% phenylmethanesulfonyl fluoride (PMSF, Sigma, USA) and protease inhibitors. Total proteins were collected and centrifuged with 20 800g (4 °C; 15 min). The protein concentration of each sample was quantified with ultraviolet spectrophotometry (Molecular Devices, USA) using the bicinchoninic acid protein assay kit (BCA, Beyotime Institute of Biotechnology, China). Equal amounts (30 μg) of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto polyvinylidene fluoride (PVDF, GE Healthcare, USA) transfer membranes. The PVDF membranes were blocked with 5% milk or 3% BSA in Tris-buffered saline and Tween 20 (TBST, 0.05% Tween 20, 20 mmol L⁻¹ Tris–HCl, 140 mmol L⁻¹ NaCl) at 37 °C for 2 h, incubated with primary antibodies in 3% BSA–TBST (4 °C, overnight), washed three times with TBST, and incubated with the appropriate HRP-labeled secondary antibodies in 1% BSA–TBST (2 h, 37 °C). After the membranes were washed five times with TBST, the protein bands of interest were detected with enhanced chemiluminescence (ECL, Beyotime Institute of Biotechnology, China). Images of the bands were analyzed with Molecular Image® ChemiDoc™ XRS+ with Image Lab™ Software (Bio-Rad Laboratories, Inc., USA). The tests were performed at least three times.

The mechanism by which the SV-peptide regulates the migration of SK-OV-3 cells was further investigated with immunofluorescence staining of the RhoA and Rac1 GTPases. In brief, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, USA), permeabilized with 0.5% Triton X-100 (10 min), and blocked by adding 1% (w/v) BSA (15 min, at room temperature). They were incubated with primary antibodies (1 : 100 dilution, Santa Cruz™, USA) at 4 °C overnight, followed by the appropriate secondary FTIC-conjugated IgG (Goat anti-mouse IgG, green color fluorescence) (60 min at 37 °C). Each step was followed by washing twice with PBS for 5 min. The nuclei were stained by 30 min incubation with Hoechst 33258. After washing twice with PBS, the cells were observed using laser scanning confocal microscopy (Leica TCS SP5, Germany). The F-actin filaments were stained by fixing the cells in Alexa Fluor 488 phalloidin (10 min, room temperature), without secondary antibody. The following steps were as mentioned above.

3.5 Cell viability assay–MTT assay

The MTT assay was used to test the effect of the SV-peptide on cell viability. Cell suspensions (200 μL per well, 1 × 10⁴ cells, 6 wells) were dispensed into 96-well plates (BD, USA) and incubated overnight. The culture medium was replaced with 200 μL PBS (control cells) or SV-peptide (0.5, 0.1, 0.05, 0.025, 0.01, 0.005, 0.0025, and 0.001 mg mL⁻¹ PBS). Three identical plates were cultured 24, 48 and 72 h (5% CO₂; 37 °C), then 20 μL MTT–PBS (5 mg mL⁻¹) was added to each well. After 4 h incubation at 37 °C, the MTT–PBS solution was replaced by 150 μL dimethyl sulfoxide (DMSO, Sigma, USA). After an incubation of 30 min at room temperature, the absorbance of solubilized formazan was measured at 490 nm with a microplate reader (Molecular Devices, Versa Max, USA). Cell viability was calculated using the formula: cell viability (%) = optical density (OD) of treated cells/OD of control cells. All experiments were performed in triplicate.

3.6 Cell structure examination by transmission electron microscopy

The cytotoxicity of the SV-peptide was investigated by examining the ultrastructure of cell SK-OV-3, A2780, A549, and HUVEC using transmission electron microscopy (TEM, JEOL-200, Japan). Cells exposed to 0.1 mg mL⁻¹ SV-peptide for designated periods were washed three times with PBS and fixed with Karnovsky's EM fixative (2.5% glutaraldehyde and 2% paraformaldehyde in 80 mM phosphate buffer, pH 7.3–7.4). Secondary fixation was done in 1% osmium tetroxide with 1.5% potassium ferrocyanide in double distilled H₂O (1 h, 4 °C). Dehydration was performed using graded concentrations of ethanol with three changes at 100%. Pure Epon–Araldite resin without methyl anhydride was allowed to infiltrate the cells overnight at room temperature. The next day, excess resin was removed and fresh resin was added to the appropriate depth. The sample was polymerized (18 h) and ultrathin sections were cut en face (parallel to the surface of the culture) using a Leica Ultracut UCT ultramicrotome (MT-X; RMC Inc., Tucson, AZ, Germany). They were stained with uranyl acetate and lead citrate before viewing on TEM.

3.7 Effect of the SV-peptide on metalloproteinases

The protein expression levels of MMP-2 and MMP-9 were detected with the ELISA assay. In brief, SK-OV-3 cell suspensions (200 μL, 1 × 10⁴ cells, 6 wells) were dispensed into 96-well plates (BD, USA) and incubated overnight. The culture medium was replaced with 200 μL PBS (control cells) or 200 μL SV-peptide/PBS (0.0, 0.5, 0.1, 0.02, and 0.004 mg mL⁻¹), then the plate was cultured at 37 °C for 24 and 48 h. The MMP concentrations were quantified using ELISA assay kits, as recommended by the manufacturer (R&D, USA).

3.8 Statistical analysis

Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) version 13.0 software. All data were obtained in triplicate and reported as means ± standard deviations (SD). Multiple group comparisons were conducted by one-way analysis of variance (ANOVA), followed by Tukey's post hoc test. In all statistical evaluations, p < 0.05 was considered as statistically significant.

4. Conclusions

The study suggests that the SV-peptide may significantly reduce tumor growth and invasiveness. Furthermore, we provided evidence that the SV-peptide may inhibit cancer cell migration through inhibition of FAK–Rho signaling and downregulate MMP-2 and MMP-9 expression. While the mechanism remains unclear, it is tempting to speculate that the PSP motif may bind to proteins affecting critical signaling events.¹⁶ For instance, there are SH2 domains in the p85 subunit of PI3K, which bind and activate FAK.³⁵ On the other hand, the PSP motif of the SV-peptide binds to SH3 domains through prolines. Therefore, the SV-peptides may bind to FAK on their SH3 domains and prevent PI3K-mediated activation. Because the SV-peptide recognizes normal and cancer cells, applications in anti-cancer drug targeting delivery are not envisioned. Nonetheless, the potent effects on cancer cell viability and migration support a therapeutic potential for tumor suppression.

Acknowledgements

This work has been supported by the Natural Science Foundation of China (No. 51503140, 5140227, 11502158), the support of the Scientific and Technological Innovation Programs of Higher Education Institutions in Shanxi (2013111, 2015140), Natural Science Foundation of Shanxi Province (No. 2013021014-2, 2015021195) is also acknowledged with gratitude.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra08755d

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