Tamarixetin Suppresses Colorectal Cancer Progression by Targeting DPP7-Mediated WNT3A/β-Catenin Signalling Pathway

Abstract

Colorectal cancer (CRC) patients have had limited benefits from conventional chemotherapy, highlighting the need for improved therapeutic strategies. Natural compounds have emerged as promising alternatives due to their potent anti-cancer properties and reduced side effects. Tamarixetin is an O-methylated flavonol derived from Azadirachta indica, but its potential and clinical utility to suppress CRC progression remain unknown. To figure out the underlying mechanism, the inhibitory effects of Tamarixetin on CRC were evaluated by in vitro assays; the validation of Tamarixetin-mediated tumour suppression was performed with CRC xenografts and patient-derived organoids. Our results demonstrated that Tamarixetin significantly reduced the proliferation of CRC cells (HT-29 and HCT-116) in a dose-dependent manner, with minimal effects on normal colonic epithelial cells (NCM460). Furthermore, Tamarixetin inhibited proliferation, migration, and invasion of CRC cells, leading to reduced xenograft tumour growth and sensitising CRC to Oxaliplatin. Mechanistically, The expression and protein levels of DPP7 in CRC cells were suppressed by Tamarixetin, which lead to the downregulation of WNT3A/β-catenin signalling pathway. This study highlights Tamarixetin as a promising natural compound for CRC treatment by interfering with DPP7-mediated WNT3A/β-catenin signalling pathway. These findings provide a novel therapeutic strategy to improve outcomes of CRC.

Keywords: DPP7; Tamarixetin; WNT3A/β‐catenin Signalling pathway; anticancer therapy; colorectal cancer.

FIGURE 1

Tamarixetin inhibits the proliferation, invasion, and migration of CRC cells. (A) Chemical structure of Tamarixetin. (B) NCM460, HT‐29 and HCT‐116 cells were treated with Tamarixetin (0, 50, 100 and 150 μM) for 24 h, and then cell viability was determined by MTT assay, N = 5. (C) (Left) HT‐29 and HCT‐116 cells were treated with Tamarixetin (0, 50, 100 and 150 μM) for 24 h, and then EdU assay was used to detect cell proliferation. (Right) Corresponding statistical bar graph for EdU assay, N = 3. (D) Representative images and quantitative analysis of CRC cell migration based on wound healing assay. (E) The effect of Tamarixetin on the migration and invasion of CRC cells, N = 3. *p < 0.05; **p < 0.01; ***p < 0.001 versus non‐treated cells.

FIGURE 2

Tamarixetin inhibits the proliferation, invasion, migration and promotes apoptosis of CRC cells. (A) (Left) Nude mice bearing HT‐29 and HCT‐116 tumour of control and Tamarixetin‐treated groups. (Right) Tumour volume was monitored to measure the tumour growth in vivo. (B) Tamarixetin showed no significant effects on organ functions including liver, kidney, and heart after 18‐day administration analysing by HE staining, N = 3. *p < 0.05; **p < 0.01; ***p < 0.001 versus non‐treated cells. (C) The representative image of CRC PDOs under the treatment of Tamarixetin (100 μM) and Oxaliplatin (5 μM) for 24 h. (D) MTT assay was used to detect the cell viability of each treatment group. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 3

Tamarixetin modulates the progression of CRC through regulation of the DPP7. (A) Venn diagram depicting the intersection of potential target genes of Tamarixetin from the SuperPred database, differentially expressed genes between CRC and normal tissues from the TCGA‐COAD database, and prognosis‐related genes in CRC from the TCGA dataset. (B) qPCR analysis of four identified genes. (C) qPCR analysis of DPP7 mRNA expression in NCM460, HT‐29, and HCT‐116 cell lines. (D) Western blot analysis of the expression of DPP7 protein in NCM460, HT‐29 and HCT‐116 cell lines. (E) The effect of Tamarixetin on the protein level of DPP7 in CRC cells, GAPDH was used as a loading control, N = 3. *p < 0.05; **p < 0.01; ***p < 0.001 versus non‐treated cells.

FIGURE 4

DPP7 overexpression promotes CRC progression. (A) Transfection efficiency was detected by Western blot. (B) HT‐29 and HCT‐116 cells were DPP7 overexpressed or DPP7 overexpressed while treated with Tamarixetin (100 μM) for 24 h, and then EdU assay was used to detect cell proliferation. (C, D) The effect of DPP7 overexpression or DPP7 overexpression while treated with Tamarixetin (100 μM) for 24 h on the migration and invasion of CRC cells based on wound healing assay (C) and transwell assay (D), N = 3. *p < 0.05; **p < 0.01; ***p < 0.001 versus non‐treated cells.

FIGURE 5

DPP7 silencing inhibits CRC progression. (A) Knock‐down efficiency was detected by Western blot. (B) HT‐29 and HCT‐116 cells were DPP7 silence or DPP7 silence while treated with Tamarixetin (100 μM) for 24 h, and then EdU assay was used to detect cell proliferation. (C, D) The effect of DPP7 silence or DPP7 silence while treated with Tamarixetin (100 μM) for 24 h on the migration and invasion of CRC cells based on wound healing assay and transwell assay, N = 3. *p < 0.05; **p < 0.01; ***p < 0.001 versus non‐treated cells.

FIGURE 6

Tamarixetin promotes CRC progression via DPP7‐mediated activation of WNT3A/β‐catenin pathway. (A) GSEA revealing a strong association between DPP7 expression and the activation of the WNT signalling pathway in CRC samples. (B) Western blot analysis was utilised to assess the impact of DPP7 overexpression or silencing on the WNT signalling pathway. Subsequent treatments with Box5, Tamarixetin and a combination of both were also evaluated for their effects on the WNT pathway in the context of DPP7 modulation, GAPDH was used as a loading control, N = 3. *p < 0.05; **p < 0.01; ***p < 0.001 versus non‐treated cells.