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n-Butylidenephthalide Regulated Tumor Stem Cell Genes EZH2/AXL and Reduced Its Migration and Invasion in Glioblastoma

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n-Butylidenephthalide Regulated Tumor Stem Cell Genes EZH2/AXL and Reduced Its Migration and Invasion in Glioblastoma

Ssu-Yin Yen et al. Int J Mol Sci.

Abstract

Glioblastoma (GBM) is one of the most common and aggressive types of brain tumor. Due to its highly recurrent rate and poor prognosis, the overall survival time with this type of tumor is only 20-21 months. Recent knowledge suggests that its recurrence is in part due to the presence of cancer stem cells (CSCs), which display radioresistant, chemoresistant, self-renewal and tumorigenic potential. Enhancers of Zeste 2 (EZH2) and AXL receptor tyrosine kinase (AXL) are both highly expressed in GBM. Additionally, they are an essential regulator involved in CSCs maintenance, migration, invasion, epithelial-to-mesenchymal transition (EMT), stemness, metastasis and patient survival. In this study, we used a small molecule, n-butylidenephthalide (BP), to assess the anti-GBM stem-like cells potential, and then tried to find out the associated genes involved with regulation in migration and invasion. We demonstrated that BP reduced the expression of AXL and stemness related genes in a dose-dependent manner. The migratory and invasive capabilities of GBM stem-like cells could be reduced by AXL/EZH2. Finally, in the overexpression of AXL, EZH2 and Sox2 by transfection in GBM stem-like cells, we found that AXL/EZH2/TGF-ꞵ1, but not Sox2, might be a key regulator in tumor invasion, migration and EMT. These results might help in the development of a new anticancer compound and can be a target for treating GBM.

Keywords: AXL receptor tyrosine kinase; cancer stem cells (CSCs); enhancer of Zeste 2 (EZH2); glioblastoma (GBM); n-butylidenephthalide (BP).

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Characterization of glioblastoma (GBM) stem-like cells and GBM cells. (A) The CD133 percentage of 0XM and 1XM analyzed by flow cytometry; (B) CD133, CD44 and Nestin expression in 0XM (low CD133+) and 1XM (high CD133+); (C) Distinction of some markers in GBM cells, 0XM (low CD133+) and 1XM (high CD133+). P2: antibody conjugated positive population; PE-A: the X axis values indicated fluorescence intensity.
Figure 2
Figure 2
(AF) The cell cytotoxicity in GBM cells DBTRG and GBM stem-like cells 1XM after n-butylidenephthalide (BP), bis-chloroethylnitrosourea (BCNU) and temozolomide (TMZ) treatment; (G) The cell cycle analysis in GBM stem-like cells 1XM after BP treatment. BP induced G2/M arrest and apoptosis in 1XM.
Figure 2
Figure 2
(AF) The cell cytotoxicity in GBM cells DBTRG and GBM stem-like cells 1XM after n-butylidenephthalide (BP), bis-chloroethylnitrosourea (BCNU) and temozolomide (TMZ) treatment; (G) The cell cycle analysis in GBM stem-like cells 1XM after BP treatment. BP induced G2/M arrest and apoptosis in 1XM.
Figure 3
Figure 3
BP regulates cancer stem cell (CSC) associated protein expression in human GBM stem-like cells. The Western blot data demonstrate that BP can downregulate CSC associated gene (A) CD133 expression; (B) Sox2, Oct4 expression and (C) EZH2 expression in a dose-dependent manner after 24-h treatment with BP. CD133, Sox2, Oct4 and EZH2 expression in a time-dependant manner with BP (D) 25 μg/mL and (E) 75 μg/mL.
Figure 4
Figure 4
BP regulates AXL expression and EMT in human GBM stem-like cells. The RT-PCR and Western blot data demonstrate that BP can downregulate AXL gene expression in GBM stem-like cells 1XM in a dose-dependent manner (A); protein expression level (B); and inhibit EMT in 24-h treatment with BP (C).
Figure 5
Figure 5
BP inhibits migration and invasion in GBM stem-like cells in a dose-dependent manner. (A) Wound healing assay of 1XM treated with BP (a) or BCNU (b) at various doses in 24-h treatment. The photographs were taken under a light microscope at a magnification of ×40; (B) The quantification of the wound healing assay; (C) Transwell assay for the invasion assay with BP at various doses in 24-h treatment. (a) 0 μg/mL; (b) 12.5 μg/mL; (c) 25 μg/mL; (d) 50 μg/mL; (e) 62.5 μg/mL; and (f) 75 μg/mL; (D) The quantification of the invasion assay; (E) BP reduced the activity of matrix metalloproteinases (MMPs) in a dose-dependent manner in 24-h treatment. * p < 0.05, ** p < 0.01 and *** p < 0.001.
Figure 5
Figure 5
BP inhibits migration and invasion in GBM stem-like cells in a dose-dependent manner. (A) Wound healing assay of 1XM treated with BP (a) or BCNU (b) at various doses in 24-h treatment. The photographs were taken under a light microscope at a magnification of ×40; (B) The quantification of the wound healing assay; (C) Transwell assay for the invasion assay with BP at various doses in 24-h treatment. (a) 0 μg/mL; (b) 12.5 μg/mL; (c) 25 μg/mL; (d) 50 μg/mL; (e) 62.5 μg/mL; and (f) 75 μg/mL; (D) The quantification of the invasion assay; (E) BP reduced the activity of matrix metalloproteinases (MMPs) in a dose-dependent manner in 24-h treatment. * p < 0.05, ** p < 0.01 and *** p < 0.001.
Figure 5
Figure 5
BP inhibits migration and invasion in GBM stem-like cells in a dose-dependent manner. (A) Wound healing assay of 1XM treated with BP (a) or BCNU (b) at various doses in 24-h treatment. The photographs were taken under a light microscope at a magnification of ×40; (B) The quantification of the wound healing assay; (C) Transwell assay for the invasion assay with BP at various doses in 24-h treatment. (a) 0 μg/mL; (b) 12.5 μg/mL; (c) 25 μg/mL; (d) 50 μg/mL; (e) 62.5 μg/mL; and (f) 75 μg/mL; (D) The quantification of the invasion assay; (E) BP reduced the activity of matrix metalloproteinases (MMPs) in a dose-dependent manner in 24-h treatment. * p < 0.05, ** p < 0.01 and *** p < 0.001.
Figure 6
Figure 6
Cell migration and CSC associated genes were reversed by AXL overexpression in 1XM. (A) The photograph of colony selection and AXL overexpression confirmed by Western blotting. Migration assay of 1XM-neo (a) and 1XM-AXL (b) performed using wound healing assay (B), with BP administered at various doses in 24-h treatment, and the quantifications as (C). (D,E) When AXL was overexpressed in 1XM, AXL, EZH2, Oct4, Sox2 and CD133 were recovered. The photographs of (A,B) were taken under a light microscope at a magnification of ×40.
Figure 7
Figure 7
The photograph of colony selection (Aa,Ba) and gene overexpression confirmed by Western blotting (Ab,Bb); (Ac,Bc) EMT associated marker TGF-β, Slug and Snail expression were recovered in 1XM-EZH2, but not in 1XM-Sox2. The photographs of (Aa,Ba) were taken under a light microscope at a magnification of ×40.
Figure 8
Figure 8
The schematic mechanism of BP treatment in GBM cells. Block arrows ↙: Pathway; T bars ⊥: Inhibition the gene expression; Gray upwards arrows ↑: Increase the phenomenon; Gray downward arrows ↓: Decrease the phenomenon.

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