(-)-Epigallocatechin-3-Gallate Inhibits Colorectal Cancer Stem Cells by Suppressing Wnt/β-Catenin Pathway - PubMed (original) (raw)

(-)-Epigallocatechin-3-Gallate Inhibits Colorectal Cancer Stem Cells by Suppressing Wnt/β-Catenin Pathway

Yue Chen et al. Nutrients. 2017.

Abstract

The beneficial effects of tea consumption on cancer prevention have been generally reported, while (-)-Epigallocatechin-3-gallate (EGCG) is the major active component from green tea. Cancer stem cells (CSCs) play a crucial role in the process of cancer development. Targeting CSCs may be an effective way for cancer intervention. However, the effects of EGCG on colorectal CSCs and the underlying mechanisms remain unclear. Spheroid formation assay was used to enrich colorectal CSCs from colorectal cancer cell lines. Immunoblotting analysis and quantitative real-time polymerase chain reaction were used to measure the alterations of critical molecules expression. Immunofluorescence staining analysis was also used to determine the expression of CD133. We revealed that EGCG inhibited the spheroid formation capability of colorectal cancer cells as well as the expression of colorectal CSC markers, along with suppression of cell proliferation and induction of apoptosis. Moreover, we illustrated that EGCG downregulated the activation of Wnt/β-catenin pathway, while upregulation of Wnt/β-catenin diminished the inhibitory effects of EGCG on colorectal CSCs. Taken together, this study suggested that EGCG could be an effective natural compound targeting colorectal CSCs through suppression of Wnt/β-catenin pathway, and thus may be a promising agent for colorectal cancer intervention.

Keywords: EGCG; Wnt/β-catenin pathway; colorectal cancer stem cells.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

Figure 1

The structure of EGCG.

Figure 2

Figure 2

Enrichment of colorectal CSCs by serum-free-medium culture. DLD-1 and SW480 cells were cultured in serum-supplied medium (SSM) and serum-free medium (SFM) for six days. (A) Cell morphology was imaged under a light microscope. (B) The protein levels of CSC markers, including CD133, CD44, ALDHA1, Nanog, and Oct4, were measured by Western blotting analysis. (C) The mRNA levels of corresponding CSC markers were examined by Quantitative real-time PCR. Data are expressed as mean ± SD of three independent experiments. ** p < 0.01 compared with control group.

Figure 3

Figure 3

EGCG inhibited the CSCs properties and Wnt/β-catenin pathway in colorectal CSCs. DLD-1 and SW480 cells were treated with different concentrations of EGCG for six days. (A) Images of DLD-1 and SW480 spheroids; (B) The protein levels of colorectal CSC markers, including CD133, CD44, ALDHA1, Nanog, and Oct4, were measured by Western blotting analysis; (C) The corresponding mRNA levels were measured by Quantitative real-time PCR; (D) The protein levels of Wnt/β-catenin pathway (p-GSK3β, GSK3β, β-catenin, and c-Myc) were determined by Western blot analysis; (E) Quantitative real-time PCR analysis for the mRNA levels of β-catenin. Data are expressed as mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01 compared with control group.

Figure 4

Figure 4

EGCG reduced cell proliferation and induced apoptosis of colorectal CSCs. DLD-1 and SW480 spheroids were treated with different concentrations of EGCG for six days. (A) Protein expression levels of PCNA and Cyclin D1 were measured by Western blot analysis; (B) Quantitative real-time PCR analysis for the detection of mRNA levels of PCNA and Cyclin D1; (C) Expression levels of apoptosis related proteins, including Bcl-2, Bax, Caspase 8, Cleaved Caspase 9, and Cleaved Caspase 3, were measured by Western blot analysis. Data are expressed as mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01 compared with control group.

Figure 5

Figure 5

EGCG diminished colorectal CSCs through suppression of Wnt/β-catenin pathway. DLD-1 and SW480 spheroids were treated with different concentrations of EGCG and LiCl for six days. (A) Western blot analysis was applied to measure the protein levels of Wnt/β-catenin pathway; (B) Immunofluorescence staining images of spheroids were obtained to determine the expression of CD133. (C–E) Western blot analysis was used to measure the protein levels of CSC markers (C), proliferation-related proteins (D), and apoptosis-related proteins (E).

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