Wilms tumor-suppressing peptide inhibits proliferation and induces apoptosis of Wilms tumor cells in vitro and in vivo

Abstract

Background: Our previous study identified a Wilms tumor-suppressing peptide (WTSP) that was upregulated in healthy children, but downregulated in children with Wilms tumor (WT). This study aimed to investigate the effect of WTSP on WT growth in vivo and in vitro.

Methods: WTSP was synthesized by solid-phase synthesis of FOMC-protected amino acids. Cell growth curve, cytotoxicity, and apoptosis of WTSP-treated human WT cell line (SK-NEP-1) were determined by cell count, Cell Counting Kit-8 assay, and flow cytometry. The expression of key proteins of four WT-associated signaling pathways was determined by real-time PCR and western blotting. The WT xenograft mouse model was established by the armpit injection of SK-NEP-1 cells. The TUNEL assay was used to detect apoptosis in mouse tumor cells.

Results: WTSP inhibited the proliferation of SK-NEP-1 cells in a dose- and time-dependent manner, and it arrested SK-NEP-1 cells in G2/M phase. WTSP-treated cells exhibited a low expression of PCNA and Bcl-2 and high expression of Bax. The expression of β-catenin was markedly changed after WTSP treatment. WTSP-treated mice had significantly smaller tumors than untreated mice.

Conclusion: Our findings indicated an anti-tumor effect of WTSP, which is correlated with Wnt/β-catenin pathway. This newly identified peptide may exert a therapeutic effect of WT in the future.

Keywords: Tumor-suppressing peptide; Wilms tumor; m/z 6455.5 Da; β-Catenin.

Fig. 1

The secondary structure and the purity of WTSP. a The secondary structure model of WTSP was forecasted and established, and the matchup between the sequence and the model was shown. b The model of WTSP was viewed from other perspectives

Fig. 2

WTSP was detected by HPLC and MS. a After synthesis, WTSP was subjected to HPLC. Pure WTSP was observed at a retention time of 8.867 min; the proportion of this peptide was 96.57%. b The evidence of identity based on mass spectral characterization

Fig. 3

WTSP inhibits SK-NEP-1 cell proliferation in vitro. a The growth curve of SK-NEP-1 cells treated with different concentrations of WTSP (0, 1 × 10⁻⁶, 1 × 10⁻⁵, 1 × 10⁻⁴, 1 × 10⁻³, or 1 × 10⁻² μmol/mL) for 7 days. b Based on the growth curve of WT cells, the doubling times of the cells at different concentrations of WTSP were calculated from Td3-4, Td3-5, and Td3-6 using the Patterson formula. c SK-NEP-1 cells (1 × 10⁵ cells/well) were treated with WTSP (0, 0.5, 1.0, 1.5, 2.0, 2.5, or 3.0 × 10⁻² μmol/mL) at various time points (24, 48, and 72 h). The cell inhibition rate was determined using a CCK-8 assay. d Growth-inhibitory effect of WTSP on SK-NEP-1 cells. The IC₅₀ and IC₈₀ were calculated at 24, 48, and 72 h after WTSP treatment. e Real-time PCR was performed to examine the mRNA expression of PCNA in SK-NEP-1 cells. The protein level of PCNA was determined by western blotting. **P < 0.01 vs control. Three independent experiments

Fig. 4

WTSP induces apoptosis and necrosis of SK-NEP-1 cells in vitro. Flow cytometric analysis of annexin-V/propidium iodide (PI)-stained SK-NEP-1 cells treated with WTSP (IC₅₀ and IC₈₀) for 48 h. The dual-parameter dot plots combining annexin-V-fluorescein isothiocyanate (FITC) and PI fluorescence show the following cell populations: lower left quadrant, viable cells (annexin-V−, PI−); lower right quadrant, apoptotic cells (annexin-V+ , PI−); upper right quadrant, apoptotic cells (annexin-V+ , PI+); and upper left quadrant, necrotic cells (annexin-V−, PI+). Three independent experiments

Fig. 5

Signaling pathways affected by WTSP in vitro. a The expression of Bax and Bcl-2 was detected by real-time PCR. b The protein levels of Bcl-2, Bax, and β-catenin were determined by western blotting. c Real-time PCR was performed to examine mRNA expression of members of the Wnt pathway (DKK1, DKK2, GSK-3α, GSK-3β and β-catenin), hedgehog pathway (Patched, Shh and Smo), TGF-β pathway (TGF-β, Smad1, Smad2, Smad4 and Smad5), and growth factor receptor pathways (EGFR, VEGFC, MAPK, and AKT). **P < 0.01 vs control. Three independent experiments

Fig. 6

WTSP inhibited WT growth in vivo. a Gross observation of SK-NEP-1 subcutaneous tumors in nude mice from the control group (n = 8) and the WTSP group (n = 8) (4 mg/kg). b The mean diameter and volume of tumors from the start of WTSP treatment. **P < 0.01 vs control

Fig. 7

Anti-tumor effect of WTSP in vivo. a H&E staining. Magnification, 10 × 10. b TUNEL assay indicated increased apoptosis in WTSP-treated tumor tissue. Magnification, 20 × 10. c Immunohistochemistry analysis of PCNA. Magnification, 20 × 10. d Immunohistochemistry analysis of Bcl-2 and Bax. Magnification, 20 × 10. e Immunohistochemistry analysis of β-catenin. Magnification, 20 × 10. Three independent experiments