To verify the inhibitory mechanism of β-catenin-designed peptides in colorectal cancer(CRC) tumors, the following experiments were performed. In vitro colony formation, Transwell assays, and flow cytometry were performed to assess the biological effects of designed peptides (F18KD, F20A4-7k, F20A4-10k, and F20A3-9k + F20A4-10k + F20A5-9k) in HT-29 cells. In vivo xenograft experiments were performed and treated with peptides. Next, tumors were subjected to Hematoxylin and eosin staining (HE), immunohistochemical, and terminal deoxynucleotidyl transferase dUTP nick end labeling staining assays to evaluate the inhibitory effect of peptides on tumors. β-Catenin levels were quantified via western blotting (WB) and quantitative real-time polymerase chain reaction, and β-catenin was located using confocal laser scanning microscopy. T-cell factor-4 (TCF-4), C-myc, and CCND1 levels were quantified via WB. Results were obtained as following. First, the peptides reduced viability, migration, and invasion; promoted apoptosis; and stabilized the S phase of HT-29 cells. Second, peptides suppressed tumor growth and downregulated the expression of CD34, vascular endothelial growth factor, and β-catenin in tumors. Furthermore, we found that peptides downregulated β-catenin expression in both the cytoplasm and nucleus; TCF-4, C-myc, and CCND1 expression was also downregulated. Notably, β-catenin-targeting peptides had a better inhibitory effect on CRC than non-β-catenin-target peptides, and a combination of peptides exerted a more potent inhibitory effect on CRC than single peptides. It suggested that β-Catenin-targeting peptides promote apoptosis in CRC tumors by inhibiting activation of the Wnt/β-catenin pathway.
Keywords: Wnt/β-catenin pathway; colorectal cancer; designed peptides; in vitro; in vivo.
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