Antimetastasis Effect of Astragalus membranaceus- Curcuma zedoaria via β-Catenin Mediated CXCR4 and EMT Signaling Pathway in HCT116

Abstract

Astragalus membranaceus and Curcuma zedoaria, two traditional Chinese medicines, are widely used together in colorectal cancer adjuvant treatment. Many different mechanisms should be involved in the benefit effect of Astragalus membranaceus and Curcuma zedoaria. In this study, we established that the combined extract from Astragalus membranaceus and Curcuma zedoaria (HQEZ) decreased the metastasis ability in colorectal cancer cells (HCT116, a cell line of colorectal carcinoma established from Homo sapiens) in vitro, and the treatment induced the downregulation of EMT signal and decreased CXCR4 expression and the level of β-catenin. Overexpression of CXCR4 and the administration of the agonist and inhibitor to β-catenin signal pathway were used to explore the mechanism of Astragalus membranaceus and Curcuma zedoaria in colorectal cancer treatment. The data demonstrated that HQEZ increased the phosphorylation of β-catenin which related to the degradation of β-catenin, and it induced the downregulation of EMT signal and CXCR4. It meant that the influence of β-catenin should be a key event in the antimetastasis effects of Astragalus membranaceus-Curcuma zedoaria in colorectal cancer model. These findings revealed the potential effect and mechanism of Astragalus membranaceus-Curcuma zedoaria in colorectal cancer treatment and provided insight for optimization of the usage.

Figure 1

HQEZ induced cell damage of HCT116 cells. (a) In CCK-8 assay, HQEZ induced HCT116 cell damage with ratio, concentration, and time. (b) HQEZ induced apoptosis of HCT116 cells after a 48 hours treatment by flow cytometry. (c) Treated HCT116 cells with HQEZ induced cell cycle arrest.

Figure 2

HQEZ decreases metastasis ability of HCT116 cells. (a) In a low concentration which did not induce apoptosis, HQEZ decreased the migration and (b) invasion of HCT116 cells ∗, p<0.05 compared with vehicle.

Figure 3

Decreased EMT signal, CXCR4 signal, and β-catenin signal are related to the inhibition of HQEZ on HCT116 cells. (a) The activation of AKT and ERK was not affected by the administration of HQEZ. (b) The administration of HQEZ inhibited the EMT signaling pathway which relates to the metastasis. (c) The administration of HQEZ reduced the expression of CXCR4, an important receptor mediating liver metastasis. (d) The administration of HQEZ induced degradation of β-catenin and the downregulation of its downstream factors. ∗, p<0.05 compared with vehicle.

Figure 4

Overexpression of CXCR4 partly rescued the HCT116 cells from HQEZ. (a) Overexpression of CXCR4 rescued the metastasis ability of HCT116 cells. (b) Overexpression of CXCR4 partly rescued the EMT signal pathway and β-catenin. (c, d) In wild-type HCT116 and CXCR4 overexpressed HCT116, administration of HQEZ did not influence the mRNA level of β-catenin.

Figure 5

Activating Wnt/β-catenin pathway rescued the level of β-catenin, EMT signaling pathway, and the metastasis ability of HCT116 cells. (a, b) A potential activator of β-catenin, WAY-262611, rescued the HCT116 cells from HQEZ. (c) The degradation of β-catenin is related to the activity of GSK 3β and the phosphorylation of β-catenin. (d) Stabilization of β-catenin was important to maintain the metastasis ability. ∗, p<0.05 compared with vehicle.

Figure 6

Astragalus, Curcuma, or mixed solution showed different effects on the HCT116 cells in vitro. (a, b) Curcuma or mixed solution but not the Astragalus decreased the invasion of HCT116. (c) Curcuma decreased CXCR4 and β-catenin whose effects were less than the mixture of Astragalus and Curcuma. ∗, p<0.05 compared with vehicle.