Dexamethasone inhibits hypoxia-induced epithelial-mesenchymal transition in colon cancer

Figure 1 Effects of dexamethasone on vascular endothelial growth factor and hypoxia-inducible factor-1α expression in HCT116 and HT29 cells under hypoxia. A and B: RNA was extracted from cells treated with DEX (100, 200 nmol/L) and/or hypoxia for 24 (A) or 72 h (B). VEGF mRNA expression was analyzed by qPCR and normalized to glyceraldehyde phosphate dehydrogenase. DEX-treated HCT116 and HT29 cells in hypoxic conditions displayed the reduction of VEGF mRNA level; C and D: HIF-1α and β-actin protein expression in cells treated with DEX (50, 100, and 200 nmol/L) and/or hypoxia for 24 h was assessed by western blot analysis. HIF-1α protein level was inhibited in HCT116 and HT29 cells treated with DEX under hypoxic conditions. The error bar represents the standard deviation. ^aP < 0.05 vs hypoxia-only group. DEX: Dexamethasone; VEGF: Vascular endothelial growth factor; HIF-1α: Hypoxia-inducible factor-1α; N: Normoxia; H: Hypoxia; qPCR: Real-time quantitative polymerase chain reaction.

Figure 2 Effects of dexamethasone on the hypoxia-induced expression of epithelial-mesenchymal transition markers. A-F: RNA was extracted from cells treated with DEX (100, 200 nmol/L) and/or hypoxia for 24 (A-C) or 72 h (D-F). Snail (A, D), Slug (B, E), and Twist (C, F) mRNA expression levels were then determined by qPCR and normalized to glyceraldehyde phosphate dehydrogenase. In hypoxic conditions, transcripts of Snail, Slug, and Twist were down-regulated in DEX-treated HCT116 and HT29 cells; G and H: E-cadherin, integrin αVβ6, and β-actin expression in cells treated with DEX (100, 200 nmol/L) and/or hypoxia for 5 (G) or 7 d (H) was assessed by Western blot analysis. Fold changes of E-cadherin and integrin αVβ6 bands were calculated after normalization to β-actin. E-cadherin was induced in HCT116 and HT29 cells treated with DEX in hypoxic conditions. Reversely, integrin αVβ6 was repressed by treated with DEX in HCT116 and HT29 cells under hypoxia. The error bar represents the standard deviation. ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001 vs hypoxia-only groups. DEX: Dexamethasone; N: Normoxia; H: Hypoxia; qPCR: Real-time quantitative polymerase chain reaction.

Figure 3 Effects of dexamethasone on cell morphology in hypoxia. Immunocytochemical staining for E-cadherin and DAPI in HCT116 (A) and HT29 (B) cells. Cells were treated with DEX (100, 200 nmol/L) and/or hypoxia for 5 (A) or 7 d (B). E-cadherin (red) and DAPI (blue) were observed by immunocytochemistry. DEX rescued E-cadherin expression and morphological changes of HCT116 and HT29 cells under hypoxia. Scale bar, 50 μm; Magnification × 200. DEX: Dexamethasone; DAPI: 4',6-diamidino-2-phenylindole.

Figure 4 Effects of dexamethasone on wound healing of colon cancer cells in hypoxia. A and B: Wound healing assay in HCT116 (A) and HT29 (B) cells treated with DEX (100, 200 nmol/L) and/or DFO (100 μmol/L) for 24 h; C and D: Effect of DEX on migration of HCT116 (C) and HT29 cells (D) was plotted as a percentage of wound closure. DEX diminished the ability to recover wounds of HCT116 and HT29 cells under hypoxic conditions. The error bar represents the standard deviation. Magnification × 40. ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001 vs DFO-only groups. DEX: Dexamethasone; DFO: Deferoxamine.

Figure 5 Effects of dexamethasone on migration and invasion of colon cancer cells in hypoxia. A and B: Transwell migration assay in HCT116 (A) and HT29 (B) cells; C and D: Matrigel invasion assay in HCT116 (C) and HT29 (D) cells. Both cells treated with or without DEX and incubated for 24 (A, B) or 48 h (C, D) in normoxic or hypoxic conditions. Cells on the upper surface of the transwells stained with Hematoxylin and Eosin were counted under a light microscope. DEX repressed migration and invasion of HCT116 and HT29 cells under hypoxia. The error bar represents the standard deviation. ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001 vs hypoxia-only groups. DEX: Dexamethasone.