The iron chelators Dp44mT and DFO inhibit TGF-β-induced epithelial-mesenchymal transition via up-regulation of N-Myc downstream-regulated gene 1 (NDRG1)

Abstract

The epithelial-mesenchymal transition (EMT) is a key step for cancer cell migration, invasion, and metastasis. Transforming growth factor-β (TGF-β) regulates the EMT and the metastasis suppressor gene, N-myc downstream-regulated gene-1 (NDRG1), could play a role in regulating the TGF-β pathway. NDRG1 expression is markedly increased after chelator-mediated iron depletion via hypoxia-inducible factor 1α-dependent and independent pathways (Le, N. T. and Richardson, D. R. (2004) Blood 104, 2967-2975). Moreover, novel iron chelators show marked and selective anti-tumor activity and are a potential new class of anti-metabolites. Considering this, the current study investigated the relationship between NDRG1 and the EMT to examine if iron chelators can inhibit the EMT via NDRG1 up-regulation. We demonstrated that TGF-β induces the EMT in HT29 and DU145 cells. Further, the chelators, desferrioxamine (DFO) and di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), inhibited the TGF-β-induced EMT by maintaining E-cadherin and β-catenin, at the cell membrane. We then established stable clones with NDRG1 overexpression and knock-down in HT29 and DU145 cells. These data showed that NDRG1 overexpression maintained membrane E-cadherin and β-catenin and inhibited TGF-β-stimulated cell migration and invasion. Conversely, NDRG1 knock-down caused morphological changes from an epithelial- to fibroblastic-like phenotype and also increased migration and invasion, demonstrating NDRG1 knockdown induced the EMT and enhanced TGF-β effects. We also investigated the mechanisms involved and showed the TGF-β/SMAD and Wnt pathways were implicated in NDRG1 regulation of E-cadherin and β-catenin expression and translocation. This study demonstrates that chelators inhibit the TGF-β-induced EMT via a process consistent with NDRG1 up-regulation and elucidates the mechanism of their activity.

FIGURE 1.

Line drawing of the chemical structures of: (A) Dp44mT, (B) Dp2mT, and (C) DFO.

FIGURE 2.

TGF-β induces characteristics of the epithelial-mesenchymal transition (EMT) in HT29 and DU145 cells. HT29 and DU145 cells were treated in the presence or absence of TGF-β (5 ng/ml) for 96 h and 48 h, respectively, to induce the EMT. A, bright field images were taken to show cell morphological changes after TGF-β treatment. Scale bars: 100 μm. B, whole cell lysates were extracted and Western blotting was performed to investigate changes to molecular markers of the EMT. Densitometric analysis is expressed relative to the loading control, β-actin. C, merged images were taken to show immunofluorescence staining of E-cadherin (red), β-catenin (red), and vimentin (green) accompanied by the cell nucleus (blue) stained by DAPI. Scale bars: 20 μm. D, HT29 and DU145 cells were pretreated for 72 h and 24 h, respectively, with TGF-β (5 ng/ml), then seeded at 100,000 cells/well and incubated for 12 h/37 °C in the presence or absence of TGF-β (5 ng/ml). Migratory cells on the bottom of the polycarbonate membrane were stained (purple blue) and quantified at A₅₆₀ after extraction according to the manufacturer's protocol (Cell Biolabs). Scale bars: 200 μm. E, cells were treated with TGF-β as described in D above, and 50,000 cells/well were seeded in the transwell chamber coated with extra-cellular matrix and incubated for 12 h/37 °C. The invasion values were reported as mean relative fluorescence unit (RFU) of quadruplicate samples. Data are typical of 3–5 experiments, and the histogram values are mean ± S.D. (3–5 experiments). ***, p < 0.001, relative to control cells without TGF-β.

FIGURE 3.

Iron chelators attenuate the TGF-β-induced EMT in HT29 and DU145 cells. Cells were pretreated in the presence or absence of TGF-β (5 ng/ml) for 72 h (HT29) or 24 h (DU145) and followed by co-incubation with either DFO (100 μm), Dp2mT (10 μm) or Dp44mT (10 μm) for another 24 h. A and C, merged immunofluorescence images demonstrate membrane E-cadherin (red) and β-catenin (red) with the cell nucleus (blue) stained by DAPI. Scale bars: 20 μm. B and D, Western analysis of NDRG1 and EMT marker expression in HT29 (B) and DU145 cells (D). Densitometric analysis is expressed relative to the loading control, β-actin. Data are typical of 3–5 experiments, and the histogram values are mean ± S.D. (3–5 experiments). ***, p < 0.001, **, p < 0.01, relative to control cells without TGF-β.

FIGURE 4.

Overexpression of NDRG1 attenuates the TGF-β-induced EMT. NDRG1 overexpressing HT29 cells (clone 2), DU145 cells (clone 7), and their empty vector control cells were treated in the presence or absence of TGF-β (5 ng/ml) for 96 h and 48 h, respectively. A, Western analysis of NDRG1 and EMT marker expression. The FLAG-tagged NDRG1 was strongly expressed in both cell types (∼45 kDa). In NDRG1-overexpressing clones, there was a marked increase in the expression of FLAG-tagged NDRG1 (at ∼45 kDa) as well as the ∼44 kDa band. Densitometric analysis is expressed relative to the loading control, β-actin. B, bright field and immunofluorescence images were taken and E-cadherin (red), β-catenin (red), and nucleus (blue) were stained as described above. Scale bars: 100 μm for bright field and 20 μm for immunofluorescence. C and D, the cell migration and invasion assays were performed as described in Fig. 2, D and E. Scale bars: 200 μm. Data were repeated for 3–5 times, and the values in histograms represent mean ± S.D. (3–5 experiments). *, relative to vector control without TGF-β; #, relative to vector control with TGF-β. **, p < 0.01; ***, p < 0.001; ###, p < 0.001.

FIGURE 5.

NDRG1 knock-down mimics the TGF-β-induced EMT. A, NDRG1 knock-down HT29 cells (clone 1), DU145 cells (clone 5), and their respective control cells transfected with scrambled control shRNA were treated with or without TGF-β (5 ng/ml) for 96 h and 48 h, respectively. The efficacy of NDRG1 knock-down and the expression of EMT markers (i.e. E-cadherin, β-catenin, and vimentin) were analyzed by Western blotting. B, bright field microscopy and immunofluorescence showed that NDRG1 knock-down mimics the EMT phenotype in both HT29 and DU145 cells. Scale bars: 100 μm for bright field and 20 μm for immunofluorescence. C and D, cell migration and invasion assays were performed as described in Fig. 2, D and E. Scale bars: 200 μm. Data were repeated 3–5 times, and the values in histograms represent mean ± S.D. (3–5 experiments). *, relative to vector control without TGF-β; #, relative to vector control with TGF-β. **, p < 0.01; ***, p < 0.001; ##, p < 0.01; ###, p < 0.001.

FIGURE 6.

SMAD/Snail/Slug and Wnt pathways are involved in the NDRG1-mediated inhibition of the EMT in DU145 cells. A, Western blotting and B, immunofluorescence staining shows that NDRG1 knock-down increases Snail and Slug expression that could be responsible for E-cadherin repression. C, NDRG1 overexpression decreases the expression of canonical TGF-β/SMAD pathway components (SMAD2 and pSMAD3) and the Wnt-responsive gene, cyclin D1, while NDRG1 knock-down increases their expression. For A and C, cells were also separately incubated in the presence or absence of TGF-β (5 ng/ml) for 48 h as a positive control. Scale bars: 20 μm. Data are typical of 3–5 experiments and the histogram values are mean ± S.D. (3–5 experiments). *, p < 0.05; **, p < 0.01; ***, p < 0.001, relative to control cells without TGF-β.

FIGURE 7.

Schematic diagram showing the role of NDRG1 in inhibiting the EMT via regulation of the TGF-β/SMAD and Wnt pathways. Iron chelators up-regulate NDRG1 expression and increased NDRG1 inhibits the canonical TGF-β/SMAD pathway through decreasing expression of SMAD2 and pSMAD3. This decreases TGF-β-induced Slug expression which is responsible for E-cadherin repression. Notably, NDRG1 knock-down results in loss of membrane E-cadherin and β-catenin and their translocation into nuclei, thus activating the Wnt pathway and the transcription of cyclin D1 that plays an important role in cell cycle progression and proliferation. Taken together, NDRG1 plays a novel role in regulating the EMT through regulation of the TGF-β and Wnt/β-catenin signaling pathways.