Galangin Inhibits Cholangiocarcinoma Cell Growth and Metastasis through Downregulation of MicroRNA-21 Expression

Abstract

Galangin, a natural flavonoid product derived from the root of galangal, is emerging as a promising anticancer agent against multiple cancers. Yet, whether it also has antitumor effects on cholangiocarcinoma (CCA) and the underlying mechanism is still unknown. Herein, we demonstrate that galangin exhibits multiple antitumor effects on CCA cells including decreases cell viability; inhibits proliferation, migration, and invasion; and induces apoptosis. Moreover, those phenotypic changes are associated with downregulated microRNA-21 (miR-21) expression. To support, overexpression of miR-21 blocks galangin-mediated antisurvival and metastasis effects on CCA cells. Mechanically, galangin increases the expression of phosphatase and tensin homolog (PTEN), a direct target of miR-21, resulting in decreased phosphorylation of AKT, a protein kinase which plays a critical role in controlling survival and apoptosis. In contrast, overexpression of miR-21 abrogates galangin-regulated PTEN expression and AKT phosphorylation. Taken together, these findings indicate that galangin inhibits CCA cell proliferation and metastasis and induces cell apoptosis through a miR-21-dependent manner, and galangin may provide a novel potential therapeutic adjuvant to treat CCA.

Figure 1

Galangin inhibits cell viability and proliferation and induces apoptosis in cholangiocarcinoma cells. (a) CCK-8 analysis of cell viability in different concentrations of galangin (0, 50, 100, 150, or 200 μM)-treated cholangiocarcinoma (CCA) cell line HCCC9810 cells for 24 hours. n = 6 independent experiments. (b) EdU analysis of cell proliferation in galangin (150 μM)-treated HCCC9810 cells for 24 hours. n = 5 independent experiments. Scale: 20 μM. FACS analysis of cell apoptosis in galangin (150 μM)-treated HCCC9810 cells (c) or TFK-1 cells (e) for 24 hours. n = 3 independent experiments. Western blot analysis of Bax, Bcl-2, cleaved caspase 3, and caspase 3 expression in galangin (150μM)-treated HCCC9810 cells (d) or TFK-1 cells (f) for 24 hours. n = 3 independent experiments. Values are given as means ± SEM. ^∗P < 0.05.

Figure 2

Galangin inhibits migration and invasion in cholangiocarcinoma cells. Matrigel-coated Transwell analysis of migration and invasion in galangin (150 μM)-treated HCCC9810 cells (a) or TFK-1 cells (c) for 24 hours. Scale: 50 μM. n = 3 independent experiments. Western blot analysis of MMP9 and Vimentin expression in galangin (150 μM)-treated HCCC9810 cells (b) or TFK-1 cells (d) for 24 hours. n = 3 independent experiments. Values are given as means ± SEM. ^∗P < 0.05.

Figure 3

Overexpression of miR-21 abrogates galangin-reduced cell proliferation and galangin-induced cell apoptosis in cholangiocarcinoma cells. HCCC9810 or TFK-1 cells were transfected with 100 nM agomir nonspecific control (NC), miR-21 agomir, antagomir NC, or miR-21 antagomir for 24 hours as indicated elsewhere. (a) HCCC9810 cells were treated with galangin (150μM) for 24 hours and harvested for real-time PCR analysis of miR-21 expression. n = 3 independent experiments. (b) HCCC9810 cells were treated with galangin (150μM) for 24 hours and harvested for EdU analysis of cell proliferation. n = 5 independent experiments. Scale: 20 μM. (c) HCCC9810 cells were transfected with 100 nM antagomir NC or miR-21 antagomir and harvested for EdU analysis of cell proliferation. n = 5 independent experiments. Scale: 20 μM. (d) Real-time PCR analysis of miR-21 expression in antagomir NC or miR-21 antagomir-transfected HCCC9810 cells. n = 3 independent experiments. FACS analysis of cell apoptosis in galangin (150 μM)-treated HCCC9810 cells (e) or TFK-1 cells (f) for 24 hours. n = 3independent experiments. Western blot analysis of Bax, Bcl-2, cleaved caspase 3 and Caspase 3 expression in galangin (150μM)-treated HCCC9810 cells (g) or TFK-1 cells (h) for 24 hours. n = 3 independent experiments. Values are given as means ± SEM. ^∗P < 0.05.

Figure 4

Overexpression of miR-21 abrogates galangin-inhibited cell migration and invasion in cholangiocarcinoma cells. HCCC9810 or TFK-1 cells were transfected with 100 nM agomir nonspecific control (NC) or miR-21 agomir for 24 hours as indicated elsewhere. Matrigel-coated Transwell analysis of migration and invasion in galangin (150 μM)-treated HCCC9810 cells (a) or TFK-1 cells (c) for 24 hours. Scale: 50 μM. n = 3 independent experiments. Western blot analysis of MMP9 and Vimentin expression in galangin (150 μM)-treated HCCC9810 cells (b) or TFK-1 cells (d) for 24 hours. n = 3 independent experiments. (e) Wound healing assay analysis of migration and invasion in galangin (150 μM)-treated HCCC9810 cells. Scale: 200 μM. n = 3 independent experiments. Values are given as means ± SEM. ^∗P < 0.05.

Figure 5

Overexpression of miR-21 increases galangin-reduced PTEN/AKT signaling in cholangiocarcinoma cells. HCCC9810 cells were transfected with 100 nM agomir nonspecific control (NC) or miR-21 agomir for 24 hours. (a) Western blot analysis of PTEN expression and phosphorylation of AKT in galangin (150 μM)-treated HCCC9810 cells for 24 hours. n = 3 independent experiments. (b) Schema of galangin reduces cholangiocarcinoma cell growth and metastasis. After galangin treatment, it decreases miR-21 expression, led to increased PTEN expression, an effect that reduces phosphorylation of AKT in cholangiocarcinoma cells, resulting in reduced cell growth and metastasis. Values are given as means ± SEM. ^∗P < 0.05.