A Novel DNA Aptamer Targeting S100P Induces Antitumor Effects in Colorectal Cancer Cells

Abstract

Colorectal cancer (CRC) is a prevalent malignancy with poor prognosis and survival. As a Ca²⁺ binding protein, S100P plays a role in calcium-dependent signal transduction pathways that involve in diverse biological processes. Our previous studies have shown that S100P is overexpressed in CRC tissues and regulates cell growth, invasion, and metastasis in CRC. Therefore, S100P is expected to be an effective target for CRC therapy. Aptamers are short single-stranded oligonucleotides that could serve as specific and high-affinity probes to a wide range of target molecules for therapeutic purposes. In this study, we generated a novel DNA aptamer against S100P (AptS100P-1) by way of the SELEX process and high-throughput sequencing. The binding assay showed that AptS100P-1 had a high affinity for S100P protein. Further experiments indicated that AptS100P-1 is relatively stable in a cell culture system and could be used in flow cytometry analysis, dot blot assay, and fluorescence microscopy analysis to detect S100P. Moreover, AptS100P-1 was capable of binding to cells and had an inhibitory effect on CRC cell growth in vitro and in vivo. Also, AptS100P-1 inhibited the migration and epithelial–mesenchymal transition of CRC cells expressing S100P. These results indicate a novel DNA aptamer targeting S100P, which might be a potential therapeutic strategy for targeting S100P against S100P-expressing CRC.

Introduction

Colorectal cancer (CRC) is one of the most common malignancies in humans [1]. Despite significant improvement in its prognosis and therapy over the past few decades, nearly one-half of colorectal patients who undergo curative surgery will relapse from metastasis [2,3]. Epithelial-to-mesenchymal transition (EMT), a critical developmental regulatory program, has been shown to play an essential role in the invasion and metastasis of CRC [4]. Therefore, the development of early diagnosis and targeted treatment may potentially be significant and helpful for patients with metastatic colorectal carcinoma.

S100P is a Ca²⁺ binding protein, and has been observed to be overexpressed in almost all human adenocarcinomas, including breast [5], pancreatic [6], colon [7], and ovarian [8] carcinomas. We previously reported that S100P is overexpressed in CRC tissues and high expression of S100P is significantly interrelated with lymph node metastasis and recurrence in CRC patients [9]. Besides, overexpression of S100P had been demonstrated to mediate tumor growth and metastasis in CRC cells. Conversely, knockdown of S100P suppresses CRC cell growth, migration, and invasion in vitro, as well as tumor growth and liver metastasis in vivo, and causes a reversion of EMT [10,11]. Extracellular addition of S100P protein also increased cancer cell growth and invasion in CRC [12] and pancreatic cancer cells [13]. These findings suggest that S100P could serve as a promising candidate for a diagnostic marker, prognostic indicator, and therapeutic target in CRC.

Aptamers are short single-stranded oligonucleotides selected from a large-capacity random oligonucleotide library in vitro using a process known as SELEX (systematic evolution of ligands by exponential enrichment) [14,15]. In the basic SELEX process, the enriched aptamers are sequenced after several rounds of selection, separation, and amplification [16]. Importantly, aptamers are promising agents that can serve as specific and high-affinity probes to a wide range of target molecules for therapeutic purpose [17,18]. Compared to conventional protein antibodies, aptamers offer several potentially significant advantages in some ways. Specifically, aptamers exhibit low immunogenicity, nontoxic, rapid tissue penetration, thermal stability, cost-effective chemical synthesis, and controllable modification [19 –23]. These advantages make aptamers promising alternative molecular probes in biomedical and clinical applications [24,25].

In this study, we generated a novel specific aptamer targeting S100P protein (AptS100P-1) by the SELEX process and showed that AptS100P-1 is relatively stable in a cell culture system and could be used in flow cytometry analysis, dot blot assay, and fluorescence microscopy analysis to detect S100P. Moreover, our study revealed that AptS100P-1 inhibited growth and migration of CRC cells expressing S100P, suggesting that AptS100P-1 may serve as a promising agent for the diagnosis and treatment of CRC expressing S100P.

Materials and Methods

Cell lines

Human CRC cell lines, including SW480, SW620, DLD-1, and HT-29, were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). They were cultured in RPMI-1640 growth medium supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA) in a 37°C incubator supplemented with 5% CO₂. All procedures performed in the study were approved by the research ethics committee of Wenzhou Medical University in accordance with the Helsinki declaration.

DNA library and primers

An initial single-stranded DNA (ssDNA) Library for SELEX was synthesized. The ssDNA aptamer library consists of 40-base randomized sequences: 5′-ATCCAGAGTGACGCAGCA-N(40)-TGGACACGGTGGCTTAGT-3′. Primers for SELEX were as follows: Cy3-5′-ATCCAGAGTGACGCAGCA-3′ (Forward primer) and biotin-5′-ACTAAGCCACCGTGTCCA-3′ (Reverse primer). The ssDNA library, PCR primers, Cy3-conjugated aptamer, and biotin-conjugated aptamer were synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China).

SELEX procedures

To select aptamers that sensitively and specifically recognize S100P, 10 μL Ni-Sepharose bead suspension (more than 2 × 10⁶ beads/tube; GE Healthcare, USA) was washed with 1 mL washing buffer (0.1% Tween-20 in Dulbecco's Phosphate Buffered Saline (DPBS), pH 7.4) thrice and resuspended the beads thoroughly in a final volume of 200 μL Binding Buffer I (1% bovine serum albumin [BSA], 0.1% Tween-20, 0.2 mg/mL tRNA, DPBS, pH7.4). Then, 10 μg recombinant human S100P protein (Rb S100P; R&D Systems, Minneapolis, MN, USA) or 6 × Histidine-tag Polypeptide (R&D Systems) was added to the resuspended Ni-Sepharose beads and incubated for 30 min at room temperature to prepare protein-coated Ni-Sepharose beads. The prepared S100P protein-coated Ni-Sepharose beads were used for positive selection, and the prepared His-tag-coated Ni-Sepharose beads were used for counter selection.

Resuspended initial ssDNA library pools (14 nmol) in 1 mL Binding Buffer I was first denatured by heating at 95°C for 5 min and slowly cooled down to room temperature for 10 min to renature. The prepared S100P protein-coated Ni-Sepharose beads were added to the renatured ssDNA library and incubated for 1 h at room temperature for positive-SELEX. After incubation, the ssDNA library was washed to remove unbound ssDNA aptamers and resuspended in 500 μL of DNase-free deionized water. The suspension was then heated at 95°C for 10 min and centrifuged at 12,000 rpm for 5 min. The supernatant, which contained the dissociated ssDNA aptamers, was collected and then amplified by PCR (95°C, 30 s; 56.3°C, 30 s; and 72°C, 30 s) using Cy3-5′-ATCCAGAGTGACGCAGCA-3′ (Forward primer) and biotin-5′-ACTAAGCCACCGTGTCCA-3′ (Reverse primer). Cy3-labeled primer was used to monitor the progress of selection by flow cytometry, and biotin-labeled primer was used to separate the single-strand aptamer by streptavidin-biotin interaction. The products were monitored by 1.5% agarose gel electrophoresis (with 1:10,000 GelRed, Biotium, USA). Streptavidin agarose beads were used to capture the biotinylated antisense strands, and the sense strands were eluted by 200 mM NaOH. The ssDNA was purified by NAP-5 desalt columns. The appropriate amount of ssDNA was used for the next round of SELEX.

To reduce nonspecific binding species, the counter selection was introduced after 2-, 3-, and 4-round positive selections. The prepared His-tag-coated Ni-Sepharose beads were added to the ssDNA library pool (the ssDNA species from the previous round of positive selection) and incubated for 30 min at room temperature. After incubation, the ssDNA species that bind to the prepared His-tag-coated Ni-Sepharose beads were removed by magnetizing beads. To increase the selection pressure, we decreased the number of S100P protein-coated Ni-Sepharose beads as well as the incubation time eventually in positive selection, but increased the His-tag-coated Ni-Sepharose beads and incubation time in counter selection. The selection process was repeated iteratively until significant affinity toward the S100P protein-coated Ni-Sepharose beads was observed by flow cytometry, and was stopped when no further progress was observed.

DNA sequencing and structure prediction

The enriched ssDNA (second, fourth, and fifth rounds) pools were amplified with adapter primers for sequencing by PCR method, respectively, and the resulting adapter-fused libraries were gel purified and subjected to next-generation sequencing (NGS) using a Miseq device and a 300-cycle paired-end sequencing protocol by Sangon Biotechnology Co., Ltd. (Shanghai, China). The data were analyzed to obtain the enrichment of abundance of top 10 ssDNA sequences in the second, fourth, and fifth rounds, and the highest abundance sequence from fifth round-enriched ssDNA pools was named AptS100P-1. The secondary structure of the selected aptamer was predicted using the MFold web service (http://unafold.rna.albany.edu/?q=mfold/DNA-Folding-Form).

Flow cytometry analysis

To monitor the binding of the enriched ssDNA pools during SELEX, the screened Cy3-labeled ssDNA pools of 2, 4, and 5 rounds or random pools were incubated with the prepared S100P protein- or His-tag-coated Ni-Sepharose beads for 1 h at room temperature. The ssDNA pools were washed with phosphate-buffered saline (PBS) twice to remove unbound ssDNA aptamers. Then, it was resuspended in 500 μL of PBS and the fluorescence intensity was analyzed by flow cytometry. The Cy3-labeled random pools served as a negative control.

To determine the binding capacity studies of the selected aptamers for S100P, the candidate Cy3-labeled aptamers were incubated with the prepared S100P protein- or His-tag-coated Ni-Sepharose beads for 1 h at room temperature. The fluorescence intensity was analyzed by flow cytometry.

To determine the cell specificity of the selected aptamers, CRC cell lines, including SW480, SW620, DLD-1, and HT29, were washed twice with cold PBS and cells were resuspended in 5% BSA solution at a concentration of 5 × 10⁵ cells/tube for 30 min. Then cells were incubated with a primary antibody or a Cy3-conjugated aptamer for 30 min. After thorough washing, cells were incubated with a fluorescence-conjugated secondary antibody for 30 min in the dark (this step should be skipped when added a Cy3-conjugated aptamer) at room temperature. Then the cells were analyzed by flow cytometry. Rabbit monoclonal IgG was used as the isotype control and the Cy3-conjugated random pools were used as negative control, respectively. All experiments for binding assays were repeated thrice.

Determination of equilibrium dissociation constant

To determine the equilibrium dissociation constant (Kd) value of the selected aptamers, we measured the binding of various concentrations of the 5′-end Cy3-labeled AptS100P-1 (0, 3.125, 6.25, 12.5, 25, 50, 100, 200, and 400 nM diluted in Binding Buffer I) with a constant amount of Rb S100P protein. The mean fluorescence density of AptS100P-1 bound to Rb S100P protein was determined using flow cytometry, and Kd was calculated by fitting the binding data to a one-site saturation equation: Y = Bmax × X/(Kd + X), in Origin Pro software 9.1 (OriginLab, Inc., Northampton, Massachusetts, USA). Bmax is the saturated binding, Y is the fluorescence intensity, and X is the concentration of the selected aptamer. The 6 × His polypeptide was used as a negative control.

Dot blot assay

The binding affinity between the selected aptamer and S100P protein was assayed by dot blot. Briefly, S100P protein (1 μg) was spotted onto a nitrocellulose membrane and dried at room temperature. After being blocked with Binding Buffer II (3% BSA, 0.1% Tween-20, 0.2 mg/mL tRNA, DPBS, pH 7.4), the membrane was incubated with renatured biotin-tagged random pools or 5′-end biotin-labeled AptS100P-1 without flanking sequences (diluted to 200 nM in PBS) at room temperature for 1 h. The membrane was then washed thrice with PBST (PBS with 0.1% Tween-20, pH7.4) and incubated with streptavidin-conjugated horseradish peroxidase (SA-HRP) (1:2000 diluted in PBST) at room temperature for 45 min, and finally, the blots were developed by enhanced chemiluminescence (ECL; Santa Cruz Biotechnology, Santa Cruz, CA, USA). An equal amount of random pools served as a control.

To confirm the affinity and specificity of the selected aptamer, the culture supernatant of DLD-1 cells, which expressed a high level of S100P protein, was concentrated by Vivaspin sample concentrators (GE Healthcare). The concentration of total protein was measured by the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), and the dot blot assay was performed.

Western blot analysis

Cells were lysed in RIPA buffer (Thermo Fisher Scientific) containing blends of protease and phosphatase inhibitors (Thermo Fisher Scientific). The concentration of total protein was measured by the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Total protein (40 μg) was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 12% gel (120 V) and subsequently transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked in 5% BSA for 2 h at room temperature and incubated with primary antibodies overnight at 4°C. Subsequently, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Antibody binding signals were detected using an ECL detection system (Bio-Rad, California, USA).

The following primary antibodies were used: rabbit anti-S100P (diluted 1:1,000; Cat. No. ab133554; Abcam, Cambridge, United Kingdom), mouse anti-GAPDH (diluted 1:4,000; Cat. No. ma5-15738; Thermo Fisher Scientific), rabbit anti-E-cadherin (diluted 1:5,000; Cat. No. 3195S; Cell Signaling Technology), and rabbit anti-Vimentin (diluted 1:5,000; Cat. No. 550513; BD). The secondary antibody was goat anti-rabbit IgG (H+L) HRP conjugated or goat anti-mouse IgG (H+L) HRP conjugated (diluted 1:5,000; Thermo Fisher Scientific).

Fluorescence microscopy analysis

Cells were grown on a coverslip and fixed in 4% paraformaldehyde at 4°C for 20 min. After blocking with 10% goat serum in PBS with 0.3% Triton X-100 solution, cells were incubated with a primary antibody or a Cy3-conjugated AptS100P-1 overnight at 4°C. After thorough washing, cells were incubated with a fluorescence-conjugated secondary antibody (Alexa-Fluor^® 594 goat anti-rabbit antibody IgG, Life Technologies) for 1 h (this step should be skipped when added a Cy3-conjugated aptamer). Nuclei were counterstained with 4′-6-diamidino-2-phenylindole (DAPI; Life Technologies) and observed and imaged using a fluorescence microscope (Olympus, Lake Success, NY). Rabbit monoclonal IgG was used as the isotype control and the Cy3-conjugated random pools were used as negative control, respectively. Experiments were repeated thrice.

To analyze the binding of AptS100P-1 to cells, DLD-1 cells were grown in six-well plates for 24 h and then incubated with Cy3-labeled aptamers (200 nM) for 8 h. The cells were washed and imaged by a fluorescence microscope.

Biological stability assay

To test the stability of aptamer in biological fluids, AptS100P-1 (2 μg) with flanking sequences was cultured with 100 μL RPMI 1640 medium supplemented with 10% FBS. The remaining levels of AptS100P-1 were detected by 1.5% agarose gel electrophoresis at 0, 2, 4, 8, 12, 24, and 36 h. Densitometry was measured using ImageJ software and the relative densitometry was graphed as fold change compared with 0-h band density. The half-life of AptS100P-1 was calculated using GraphPad Prism software.

Cell migration assays

The cell migration ability was examined using 24-well Transwell inserts with 8 μm pore filters (Costar; Corning Incorporated, Cambridge, MA, USA) according to the manufacturer's instructions. Briefly, DLD-1 (5 × 10⁴/200 μL) and SW480 cells (1.5 × 10⁵/200 μL) were seeded onto the Transwell filter membrane chambers in a medium without FBS. An equal volume of medium containing AptS100P-1 and/or Rb S100P protein was added to each well. A medium supplemented with 20% FBS was added to the lower chambers as a chemoattractant. After being incubated at 37°C for 2 days, cells in the lower chambers were fixed with 4% paraformaldehyde and then stained with 0.1% crystal violet solution. The nonmigratory cells were removed from the upper chamber surface using cotton swabs, and the migrated cells were counted in five different fields (fields were randomly selected under a microscope at magnification × 200). Experiments were repeated thrice.

Cell proliferation assays

The cell counting kit-8 (CCK-8, Dojindo, Japan) assay was used to examine cell proliferation. Cells (4,000 cells/well) were seeded in 96-well plates with a volume of 100 μL complete medium. An equal volume of medium containing AptS100P-1 and/or Rb S100P protein was added to each well. CCK8 solution (10 μL) was added to each well at 48 h. After incubation for 3 h, the absorbance value (OD) was measured at 450 nm. Experiments were repeated thrice.

In vivo xenograft treatment study

The animal experiment was approved by the Animal Experimental Ethics Committee of Wenzhou Medical University (Permit No.: wydw2019-0507). All male BALB/c nude mice (5–7 weeks old) were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd., Beijing, China. The mice were kept under laboratory conditions for 1 week before further experimentation. The xenograft model of CRC was established by subcutaneous injection of human DLD-1 CRC cells (1 × 10⁶ cells suspended in PBS 200 μL/injection) into the right flank of mice. One week after inoculation, the tumor-bearing nude mice were randomly divided into PBS control group and AptS100P-1 treatment group (n = 4 mice/group). PBS or AptS100P-1 (2.5 mg/kg, suspended in PBS 50 μL/injection) was administered intratumorally twice a week. The body weight of mice and tumor volumes were measured before injection twice a week. Tumor length and width were measured using a Vernier caliper. The volume of the tumor (mm³) was calculated using the following formula: 0.5 × length × width². After 5 ½ weeks, mice were sacrificed, and the tumors were harvested and evaluated.

Statistical analysis

Data are presented as mean ± standard deviation. Statistical analysis was performed with GraphPad Prism 7.0 (GraphPad Software, Inc., La Jolla, CA, USA). Student's t-test was used to compare values. All statistical tests were two sided. A P-value of less than 0.05 was considered to be statistically significant.

Results

Selection of aptamers specific to human S100P protein

To monitor the binding of the enriched ssDNA pools to human S100P protein during SELEX, we analyzed the binding of the enriched ssDNA pools (second, fourth, and fifth rounds) by flow cytometry. The second round of ssDNA pools showed the binding capacity to the S100P protein-coated Ni-Sepharose beads compared to the negative control (random pools) or blank beads, and the fluorescence signal gradually increased and reached a maximum after the fourth SELEX round (Fig. 1A). There was no significant difference observed between fourth and fifth ssDNA pools. In contrast, the selected pools showed no binding to the His-tag-coated Ni-Sepharose beads (Fig. 1B), indicating that the counter selection was successful.

FIG. 1.

Selection of S100P-binding DNA aptamers. (A) The binding capacity assays of the enriched ssDNA pools (second, fourth, and fifth) with the S100P protein-coated Ni-Sepharose beads were observed by flow cytometry; (B) the selected pools showed no binding to the His-tag-coated Ni-Sepharose beads; (C) the dissolution curve analysis of the enriched pools is shown; (D) the abundance of enrichment of the top 10 nucleic acid aptamer sequence. After the second, fourth, and fifth rounds of screening, the Cy3-labeled ssDNA pool was picked up and sequenced. The highest abundance aptamer sequence is named AptS100P-1. ssDNA, single-stranded DNA. Color images are available online.

As a paralleled and complementary method to affinity tests and binding analyses, melting curve analysis was drawn into monitoring the convergence of the aptamer species during selection progress [26]. Melting curves of every round of DNA pools provided further evidence of the enrichment of aptamer species. The dissolution curve analysis was used to assess the complexity of the enriched pools. As the selection round increases, analysis of DNA melting curves showed increased melt temperature, which indicated a significant drop in the library complexity (Fig. 1C). The enriched ssDNA (second, fourth, and fifth rounds) pools were picked up and subjected to NGS. The enrichment of abundance of top 10 ssDNA sequences in the second, fourth, and fifth rounds of DNA pools is shown in Fig. 1D; the highest abundance aptamer from the fifth round of DNA pools accounted for more than 10%, and the other sequences accounted for less than 2% individually in the fifth round.

Characterization of AptS100P-1 specific to human S100P

The highest abundance aptamer was chosen as a candidate aptamer after being added to the primer region at each end and named AptS100P-1, the sequence of which is 5′-ATCCAGAGTGACGCAGCACAGGACTGCTTAGGATTGCGAAGTGCATAGAGCGGCTATATGGACACGGTGGCTTAGT-3′ (Fig. 2A). As shown in Fig. 2B, the secondary structure of AptS100P-1 was predicted by MFold software and consisted of two short stem regions and a loop region. This structure may be related to the specific binding of AptS100P-1 to human S100P protein. The equilibrium dissociation constant (Kd) of the AptS100P-1 was 94.8 ± 30.1 nM, which indicated the high affinity to Rb S100P protein (Fig. 2C). The flow cytometry analysis verified that the AptS100P-1 could bind specifically to the S100P protein-coated Ni-Sepharose beads, whereas they had no or minimal reaction to the His-tag-coated Ni-Sepharose beads. As expected, the random pools as a negative control possessed no binding reaction to either S100P protein-coated Ni-Sepharose beads or His-tag-coated Ni-Sepharose beads (Fig. 2D). A further dot blot assay also confirmed that AptS100P-1 could specifically bind to S100P protein; the random pool as a negative control did not react with S100P protein (Fig. 2E). These results suggested that AptS100P-1 could specifically bind to S100P.

FIG. 2.

Characterization of AptS100P-1 binding to recombinant human S100P protein. (A) Sequence of AptS100P-1 aptamer; (B) secondary structure prediction of AptS100P-1 by the MFold software; (C) the binding of various concentrations of the 5′-end Cy3-labeled aptamer (0, 3.125, 6.25, 12.5, 25, 50, 100, 200, and 400 nM) with a constant amount of Rb S100P protein was determined; (D) AptS100P-1 binding specificity for human S100P protein was analyzed by flow cytometry; (E) AptS100P-1 binding specificity was analyzed by dot blot. An equal amount of the random pool was used as a control. Color images are available online.

Specific recognition of S100P-expressing CRC cells by S100P aptamer (AptS100P-1)

The S100P protein expression levels in CRC cell lines SW480, SW620, DLD-1, and HT29 were determined by western blotting. As shown in Fig. 3A and B, SW480 cell line exhibited a very low expression level of S100P; however, SW620, DLD-1, and HT-29 cell lines showed a relatively high expression level of S100P. To explore the specific recognition of S100P-expressing CRC cells by AptS100P-1, we detected S100P expression among CRC cell lines with different S100P expression levels using immunofluorescent staining and flow cytometry with a commercial specific ani-S100P antibody and AptS100P-1, respectively. From the immunofluorescent staining analysis, we could observe that the S100P low-expression cell line SW480 has a very low fluorescence signal and the S100P high-expression cell lines SW620, DLD-1, and HT-29 showed a relatively high fluorescence signal both using ani-S100P antibody and AptS100P-1, respectively (Fig. 3C). A further flow cytometry assay quantitatively measured the binding of AptS100P-1 in cells. We also observed that AptS100P-1 could bind to S100P protein (Fig. 3D). These results indicated that the AptS100P-1 could serve as an excellent analytical tool in detecting S100P protein expression.

FIG. 3.

Specific recognition of S100P-expressing colorectal cancer cells by S100P aptamer (AptS100P-1). (A) The expression level of S100P in colorectal cancer cell lines SW480, SW620, DLD-1, and HT29 by western blotting; (B) the summarized data are shown; (C) Cy3-conjugated aptamer (AptS100P-1) was used to probe the expression of S100P in SW480, SW620, DLD-1, and HT29 cell lines. Rabbit anti-S100P antibody was used as a positive control. DAPI Dye was used for routine nuclear staining. Scale bar = 100 μm; (D) AptS100P-1 binding specificity was analyzed by flow cytometry. Rabbit anti-S100P antibody was as a positive control. Rabbit monoclonal IgG (isotype control) or random pool was used as a negative control. ***P < 0.001. DAPI, 4′-6-diamidino-2-phenylindole. Color images are available online.

AptS100P-1 inhibits growth and migration of CRC cells expressing S100P

To further investigate the potential applications of AptS100P-1, we evaluated the use of AptS100P-1 as a probe on DLD-1 cells, which express high S100P protein. Figure 4A showed that AptS100P-1 binds specifically to the culture supernatants of DLD-1 cells. The band density plotted showed the changes in relative band intensity of AptS100P along with the incubation time (Fig. 4B), suggesting that the aptamer was stable in a cell culture system in 8 h and had a half-life of about 12 h. In addition, AptS100P-1 could bind to DLD-1 cells (Fig. 4C). To evaluate whether AptS100P-1 affects the growth and migration of CRC cells expressing S100P, DLD-1 cells were treated with AptS100P-1, and migration ability and growth were determined by a Transwell assay and CCK-8 assay. As shown in Fig. 4D, the DLD-1 cell migration was inhibited by AptS100P-1 in a dose-dependent manner. AptS100P-1 treatment also exerted a significant inhibitory effect on the growth of DLD-1 cells (Fig. 4E). Western blot analysis showed that the treatment of AptS100P-1 in DLD-1 cells caused an increase in EMT (a decrease in vimentin expression and an increase in E-cadherin expression) (Fig. 4F). These results suggested that AptS100P-1 could suppress DLD-1 cell growth, migration, and EMT.

FIG. 4.

The effects of AptS100P-1 on the growth and migration of colorectal cancer cells expressing S100P. (A) The cell supernatant protein level of S100P measured by dot blot assay. Rabbit anti-S100P monoclonal antibody served as a positive control. Horseradish peroxidase (HRP) or random pool was served as a negative control; (B) the stability of AptS100P in serum. Unmodified AptS100P-1 was incubated in 10% fetal bovine serum for 0–36 h and subjected to analysis by 1.5% agarose gel electrophoresis; (C) the binding and cellular localization of AptS100P-1 were assessed in proliferating DLD-1 cell cultures. DLD-1 cells were incubated with Cy3-labeled-AptS100P-1 (200 nM) at 37°C for 8 h and then observed by fluorescence microscopy. Scale bar = 200 μm; (D) inhibition of colorectal cancer cell migration by AptS100P-1. DLD-1 cells were treated with the indicated concentrations of AptS100P-1 (0, 10, 50, and 200 nM) for 48 h. The ability of cell migration was detected by a Transwell assay. Representative images from triplicate experiments and quantitation of migrated cells are shown. Magnification, × 200; (E) inhibition of colorectal cancer cell growth by AptS100P-1. DLD-1 cells were starved in serum-free RPMI-1640 medium for 16 h and then treated with AptS100P-1 (200 nM) or random pool (negative control) for 48 h. Cell growth was detected by CCK8 assays. Experiments were performed in triplicate and repeated thrice; (F) AptS100P-1 inhibits epithelial–mesenchymal transition of colorectal cancer cells. DLD-1 cells were starved in serum-free RPMI-1640 medium for 16 h and then treated with AptS100P-1 (200 nM) or random pool (negative control) for 48 h. The protein expression levels of vimentin and E-cadherin were determined by western blotting. GAPDH was used as a loading control. *P < 0.05, **P < 0.01, ***P < 0.001. CCK8, Cell Counting Kit-8. Color images are available online.

AptS100P-1 blocks the exogenous S100P-induced growth and migration of CRC cells

To further investigate whether AptS100P-1 could block the function of S100P, SW480 cells that expressed a very low S100P protein were treated with AptS100P-1 and/or Rb S100P protein. As shown in Fig. 5A and B, exogenous S100P increased the migration and growth of SW480 cells compared to control, whereas the growth and migration were suppressed in SW480 cells co-treated with AptS100P-1 and exogenous Rb S100P protein compared to those treated with exogenous Rb S100P protein alone, suggesting that exogenous S100P-induced growth and migration could be reversed by AptS100P-1 treatment. Moreover, western blot analysis showed that treatment of exogenous S100P protein induced SW480 cell EMT (an increase in vimentin expression and a decrease in E-cadherin expression), whereas SW480 cells co-treated with AptS100P-1 and exogenous Rb S100P protein showed an increase in EMT (a decrease in vimentin expression and an increase in E-cadherin expression) in SW480 cells compared to those treated with exogenous Rb S100P protein alone (Fig. 5C). These results indicated that treatment of AptS100P-1 could reverse the S100P-induced SW480 cell growth, migration, and EMT.

FIG. 5.

AptS100P-1 blocks the exogenous S100P protein-induced colorectal cancer cell growth and migration. (A) SW480 was treated with recombinant human S100P protein and the indicated concentrations of AptS100P-1 (0, 10, 200, and 500 nM) for 48 h. The ability of cell migration was determined by a Transwell assay. Representative images from triplicate experiments and quantitation of migrated cells are shown. Magnification, × 200; (B) SW480 cells were starved in serum-free RPMI-1640 medium for 16 h and then treated with recombinant human S100P protein (100 nM) and/or AptS100P-1 (200 nM) for 48 h. Inhibition effects of AptS100P-1 on the cell growth of SW480 cells were measured by CCK8 assays. Experiments were performed in triplicate and repeated thrice; (C) AptS100P-1 reversed exogenous S100P protein induced the epithelial–mesenchymal transition of colorectal cancer cells. SW480 cells were starved in serum-free RPMI-1640 medium for 16 h and then treated with Rb S100P (100 nM) and/or AptS100P-1 (200 nM) for 48 h. The protein expression levels of vimentin and E-cadherin were determined by western blotting. GAPDH as a loading control. *P < 0.05, **P < 0.01, ***P < 0.001. Color images are available online.

AptS100P-1 inhibits tumor growth in a colorectal tumor xenograft model in vivo

To further analyze the effectiveness of AptS100P-1 in vivo, we established a colorectal tumor xenograft model by subcutaneously implanting DLD-1 cells into BALB/c nude mice. Tumor growth curves showed that the AptS100P-1 treatment group effectively decreased tumor size and weight in vivo compared to the PBS control group (Fig. 6A–C). Moreover, western blot analysis verified that the treatment of AptS100P-1 caused an increase in E-cadherin expression and a decrease in vimentin expression in tumors compared to the PBS control group (Fig. 6D, E).

FIG. 6.

AptS100P-1 suppresses tumor growth in vivo. (A) Tumors dissected out from the PBS buffer control or AptS100P-1-treated groups (n = 4 per group) are shown; (B) xenograft tumor growth curves; (C) the weight of the tumor was measured; (D) The tumor tissue protein expression levels of E-cadherin and vimentin were determined by western blotting. GAPDH as a loading control; (E) The summarized data are shown. *P < 0.05, **P < 0.01, ***P < 0.001. Color images are available online.

Discussion

In this study, we reported, for the first time, a new S100P antagonistic DNA aptamer (AptS100P-1) with potential antitumor effects. AptS100P-1 was developed from the SELEX process and proved to possess good binding affinity to the S100P-expressing CRC cells in vitro. More fascinating, AptS100P-1 was functionally validated in both cells and animal models.

S100P is a 95 amino acid residue protein that belongs to a member of the S100 family, which is widely involved in the regulation of numerous cellular processes [27]. Our earlier report indicated that S100P increased CRC cell proliferation and migration in vitro and in vivo [7,9,10]. Moreover, there is extensive evidence demonstrating that S100P was found to be overexpressed in many cancer tissues such as breast, pancreatic, colon, and lung carcinomas, suggesting S100P plays an essential role in the genesis and development of many human malignant tumors [5 –10].

In this study, we used an efficient aptamer selection method [16]. The Ni-Sepharose beads were coupled with recombinant human S100P protein, and after several SELEX cycles, the S100P-specific ssDNA sequences were enriched (Fig. 1). Presumably, more SELEX cycles may further enrich DNA aptamers with high binding affinity to the protein. Also, this strategy could be easily adapted to screen for aptamers specific to a wide range of target molecules. Functional DNA or RNA aptamers usually contain particular structures [18]. Therefore, the structure of AptS100P-1 was predicted by MFold web programs. As shown in Fig. 2, the secondary structure of AptS100P-1 consisted of two short stem regions and a loop region. This structure may be related to the specific binding of AptS100P-1 to human S100P protein, but further experiments are needed.

The AptS100P-1 verified in this study has a good binding affinity and fair serum half-life without further modification. It was reported that suppressing the expression of S100P inhibits tumor cell proliferation, migration, and invasion in vitro and in vivo [10]. Consistent with previous studies, we demonstrated that the treatment of AptS100P-1 could significantly reduce CRC proliferation and migration. Moreover, we confirmed that after AptS100P-1 treatment, the protein expression levels of EMT consistently markedly decreased, implicating that AptS100P-1 should attenuate the migration by reversing the EMT process in CRC cells.

Currently, several studies have focused on the therapeutic application of aptamers [28]. The first FDA-approved aptamer drug was an RNA aptamer named Macugen, targeting vascular endothelial growth factor [29]. Several novel aptamer-based therapeutics are also in clinical trials currently [30]. In summary, our data support the translational development of AptS100P-1 with therapeutic applications in CRC. However, our animal experiments are still very preliminary for making the conclusions. Further studies are needed to clarify the toxicity and therapeutic efficacy of AptS100P-1 in animal models. Moreover, extensive optimization and modification may be required to improve its therapeutic efficacy in vivo. Also, it will be important to compare the effect of anti-S100 aptamers with antiS100 antibodies in vivo. Furthermore, further research is necessary to determine the function of AptS100P-1 in diagnosis and therapy of diseases.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported by the Medicine and Health Technology Program of Zhejiang Province [grant no. 2021426080], the National Natural Science Foundation of China [grant no. 81672385], Natural Science Foundation of Zhejiang Province [grant no. LY19H160024], and Wenzhou Science and technological Project [grant no. Y20180081].

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