SH-SY5Y was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in RPMI-1640 medium with 2 mM L-glutamine and 10% FBS. Cells were cultured at 37°C in a humidified incubator containing 5% CO₂. The medium, L-glutamine, and FBS were purchased from Sangong Biotech (Shanghai, China).

All the synthetic products were purchased from LifeTein (Beijing, China). Solid phase peptide synthesis technology was used from carboxy to amino terminus. The TP was synthesized with 55 amino acids from the C-terminus of proapoC-I and the TP labeled with FITC at the amino terminus. The signal peptide (SP) contained 26 residues from the N-terminus of proapoC-I. Peptides were isolated to a purity >95% and identified by HPLC, and sequence information was confirmed by mass spectrometry.

The Cell Counting Kit-8 (CCK-8; Dojindo, Tokyo, Japan) was used to study cell viability according to the manufacturer's instructions. A cell suspension was inoculated into a 96-well plate (3,000 cells/well), and incubated overnight. The next day, the medium was replaced by new medium containing the peptide at different concentrations (0, 0.1, 0.5, 1.0, 1.5 and 20 mg/ml) and incubated for 24, 48 and 72 h. The cells cultured with TP and SP were in the same plate. At every time-point, 10 µl CCK-8 was added to each well and the plate was incubated for 3 h at 37°C and 5% CO₂. Absorbance was measured at 450 nm using a microplate reader. The assay was performed with three replicates for each group and repeated at least three times.

The wound assay was performed to determine the cell migration capability. The cells were inoculated into 6-well plates and incubated overnight. The next day, three parallel straight lines were generated using a 200-µl pipette tip. After rinsing three times with serum-free medium, the cells were incubated in serum-free medium with various TP concentrations (0, 0.1 and 0.5 mg/ml) for 24 h. The wound healing area was photographed and analyzed by IPP software. The relative migration was calculated as a percentage of the area covered by the migrated cells compared to the original wound area.

Migration and invasion assays were performed using 24-well Transwell cell culture chambers (Corning, NY, USA) or Matrigel coated invasion chambers. The cells (2×10⁴ for migration and 4×10⁴ for invasion) were seeded into the upper chamber. The next day, plates were rinsed in serum-free medium three times and the medium was replaced with fresh medium with TP at various concentrations (0, 0.1 and 0.5 mg/ml). Medium containing 10% FBS was placed in the lower chamber. After 24 h of incubation, the cells in the upper layer of the membrane were removed with a cotton-tipped swab and cells migrated to the lower layer were fixed and stained with 0.1% crystal violet. Cells were photographed and counted in six random microscopic fields.

Cell Apoptosis (Annexin V-FITC/PI) and Cell Cycle (PI) Detection kits were purchased from KeyGen Biotech (Nanjing, China). The cells were inoculated into 6-well plates and incubated overnight. The medium was replaced by fresh medium with various TP concentrations (0, 1.0 and 1.5 mg/ml) the next day and incubated for 24 h. Cells were then harvested and analyzed using a FACSCalibur cytometer (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions. CFlow and FlowJo software was used for data analysis.

A mitochondrial membrane potential assay kit was purchased from Beyotime (Haimen, China). The cells were treated as described for the cell apoptosis and cell cycle assays and then collected and stained with JC-1 working solution at 37°C for 20 min according to the manufacturer's instructions. FlowJo software was used to analyze the results by flow cytometry.

The cells were incubated with TP (1.5 mg/ml) labeled with FITC for 24 h, followed by incubation with β-tubulin rabbit antibody at 4°C overnight and then donkey anti-rabbit IgG conjugated with Cy3 at room temperature for 2 h (Sangong, Shanghai, China). Finally, the cells were stained with DAPI working solution at 37°C for 5 min, and the distribution of peptide in the cells was observed under fluorescence microscope (Olympus, Tokyo, Japan).

A Cell Death Detection kit was purchased from Roche (Mannheim, Germany). The cells were inoculated into 96-well plates (2×10³ cells/well) and incubated overnight. The medium was replaced with fresh medium with various TP concentrations (0, 1.0, 1.5, and 2.0 mg/ml) the next day. After 24 h, the cells were fixed and incubated with TUNEL working solution at 37°C for 60 min followed by counterstaining with DAPI working solution at 37°C for 5 min. The results were observed under a fluorescence microscope.

The cells were incubated with various TP concentrations (0, 0.5, 1.0 and 1.5 mg/ml) for 24 h and collected into centrifuge tubes. After washing the cells with cold PBS twice, the proteins were extracted from the whole cell lysate by RIPA buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 1% Triton X-100, 10 mM NaF and 1 mM PMSF) containing a protease inhibitor cocktail (Sangong). Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. After blocking, the membranes were incubated with primary antibodies overnight at 4°C, including anti-Bcl-2, anti-Bcl-xl, anti-Bax, anti-Bim, anti-tBid, anti-caspase-8, anti-caspase-9, anti-caspase-3, anti-PARP, anti-cleaved-PARP, and anti-β-actin, and then incubated with anti-mouse or rabbit IgG conjugated with horseradish peroxidase at room temperature for 2 h. All the antibodies were purchased from CST (Beverly, MA, USA). The ECL western blot detection kit (Proteintech, Wuhan, China) was used for chemiluminescent visualization. β-actin was used as a loading control for whole cell extracts.

The 4-week-old BALB/C nude mice were purchased from Vital River (Beijing, China) and maintained at the SPF laboratory animal room. A total of 5×10⁶ SH-SY5Y cells in 0.1 ml of PBS were injected subcutaneously into the left sides of the dorsal area. After 2 weeks, mice bearing tumors of similar sizes were randomly divided into two groups: a PBS control group and a TP-treated group (100 mg/kg by intraperitoneal injection once daily for 21 days). After the start of treatment, tumor volume was measured and calculated every 7 days using the formula A × B²/2, where A is the longest diameter and B is the shortest. At the end of the treatment, all mice were sacrificed. Tumors were harvested, weighed and photographed. The present study was approved by the Ethics Committee of Zhengzhou University.

Tumors were fixed and embedded with paraffin. The SP immunohistochemistry method was used to assay protein expression. The results were analyzed using IPP software. Primary anti-rabbit antibodies (Bax, Bcl-2, Ki67 and PCNA) and secondary goat anti-rabbit IgG conjugated with biotin and streptavidin conjugated with HRP were purchased from Abcam (Cambridge, UK).

Statistical analysis was performed using SPSS 21.0 software. The data were expressed as the mean ± SD. The Student's t-test was used for comparisons between two groups of quantitative data. The one-way ANOVA test was used for multigroup comparisons. P<0.05 was considered statistically significant.

The proapoC-I has 83 amino acids including the SP (Met1-Gly26) and mature peptide (Thr27-Ser83). The loss of a threonine and a proline from the N-terminus of the mature peptide results in the TP (7). The sequence information is shown in Fig. 1A. In addition to what is reported in the literature, we found that the site between G26 and T27 is the cleavage site from signal to mature peptide using a signal peptide prediction website (http://www.cbs.dtu.dk/services/SignalP) (Fig. 1B). DNAStar software was used to further analyze sequence activity. Regions (above the baseline) with high hydrophilicity, antigen index, and surface probability were mainly found in the TP, suggesting that the TP is a potentially active peptide with high hydrophilicity, antigenic index, and surface probability compared with the SP (Fig. 1C). Therefore, we synthesized the TP as experimental group and the SP as control group used for CCK-8 assay.

For the TP group, increasing concentrations were correlated with the inhibition of cell proliferation at 24, 48 and 72 h (all P-values <0.001). At 24 h, the groups treated with concentrations of 0.1 and 0.5 mg/ml showed no statistically significant differences from the control group (0 mg/ml) (Fig. 1D). In the SP group, cell proliferation was not affected by different SP concentrations at 24 and 48 h (all P-values >0.05). However, at 72 h, various SP concentrations inhibited cell proliferation to some degree (P=0.001) (Fig. 1E).

In the wound healing assay, TP at 0.1 and 0.5 mg/ml inhibited the wound healing ability at 12 h compared with that in the control group (P=0.03 and P<0.001). At 24 h, the wound healing ability was also inhibited by 0.1 and 0.5 mg/ml TP (P=0.012 and P<0.001) (Fig. 2A). In the Transwell migration assay, the migration ability was reduced to 82.7 and 25.1% in response to increasing TP concentrations compared with the values in the control group (P=0.01 and P<0.001). In the invasion assay, the invasion ability was reduced to 50.4 and 25.6% compared with that in the control group (all P-values <0.001) (Fig. 2B).

In the cell apoptosis assay, increasing TP concentrations resulted in an increase in the rates of apoptosis by 22.5 and 40% (all P-values <0.01, Fig. 3A). The percentage of cells in S phase increased to 35.9% (P>0.05) and 42.6% (P=0.03) compared with that in the control group at 27.1%, and the percentage of cells in G2/M decreased to 25.9% (P>0.05) and 19.8% (P=0.04) compared with that in the control group at 29.9% (Fig. 3B). In the mitochondrial membrane potential assay, increasing TP concentrations increased the percentage of cells with JC-1 monomers to 33.7 and 58.8% (all P-values <0.001, Fig. 3C).

Incubation with FITC labeled TP at 1.5 mg/ml for 24 h resulted in the entry of the peptide into the cells. The result showed that the TP was mainly concentrated in the cytoplasm and cell membrane, with a fraction detected in the nucleus (Fig. 4A).

In the TUNEL assay, increasing TP concentrations increased DNA cleavage during apoptosis. The results showed that the survival rate of the cells was reduced to 75% (P=0.011), 46.7% (P=0.009), and 23.5% (P=0.002) in response to increasing TP concentrations (Fig. 4B). These data provided direct evidence that the TP induced apoptosis.

Increasing TP concentrations resulted in the downregulation of Bcl-2 and Bcl-xl which are anti-apoptotic proteins of the Bcl-2 family. Downregulation of Bcl-2 or Bcl-xl affects mitochondrial function. Upregulation of Bax causes the release of cytochrome c through oligomerization from the cytosol to the mitochondrial outer membrane. Bim accelerates this process by inhibiting anti-apoptotic proteins such as Bcl-2 or Bcl-xl. The release of cytochrome c activates caspase-9, and cleaved caspase-9 further activates caspase-3. Finally PARP is cleaved into its active form resulting in DNA damage. Disruption of mitochondrial function by the intrinsic apoptosis pathway was observed. On the other hand, activation of caspase-8 is most likely associated with the stimulation of cell surface death receptors such as tumor necrosis factor receptors (TNF-R). Active caspase-8 directly cleaves caspase-3 to induce apoptosis. Furthermore, Bid is cleaved by active caspase-8 into tBid, which becomes an integral membrane protein in mitochondria and recruits Bax from the cytosol to the outer membrane, eventually causing the release of cytochrome c. The results showed that the TP induced apoptosis through the intrinsic and extrinsic pathways (Fig. 5).

Tumor volume was measured every 7 days in a PBS control group and a TP-treated group (100 mg/kg by intraperitoneal injection once daily for 21 days). After 7 days, the tumors in the control group began to grow rapidly. Measurements at 14 days showed a significantly greater tumor volume in the control group than in the TP group (P=0.005) (Fig. 6A). Mice were sacrificed at the end of treatment after 21 days. Tumors were harvested, weighed and photographed. The results showed that the tumor volume (P=0.017) and weight (P=0.004) were reduced in the TP group (Fig. 6A-C). Tumors were fixed and embedded with paraffin, and the SP immunohistochemistry method was used to examine relative protein expression (Fig. 6D). Downregulation of PCNA and Ki67 in the TP group indicated the inhibition of tumor cell proliferation. Upregulation of Bax and downregulation of Bcl-2 probably enhanced the permeabilization of the mitochondrial membrane. The release of cytochrome c eventually induced apoptosis in the tumor.