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Copper depletion inhibits CoCl2-induced aggressive phenotype of MCF-7 cells via downregulation of HIF-1 and inhibition of Snail/Twist-mediated epithelial-mesenchymal transition.

Li S, Zhang J, Yang H, Wu C, Dang X, Liu Y - Sci Rep (2015)

Bottom Line: Using CoCl2-induced EMT of human breast carcinoma MCF-7 cells, we found that TEPA, a copper chelator, inhibited EMT-like cell morphology and cytoskeleton arrangement triggered by CoCl2; decreased the expression of vimentin and fibronectin, markers typical of EMT; inhibited HIF-1 activation and HIF1-α accumulation in nuclear; and down-regulated the expression of hypoxia-associated transcription factors, Snail and Twist1.Immunohistochemical analysis of the xenograft further demonstrated that TEPA administration significantly inhibited tumor angiogenesis, down-regulated hypoxia-induced transcription factors, Snail and Twist1, leading to decreased transactivation of EMT-associated marker genes, vimentin and fibronectin.These results indicate that TEPA inhibits CoCl2-induced EMT most likely via HIF1-α-Snail/Twist signaling pathway, and copper depletion may be exploited as a therapeutic for breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics, School of Life Science and Technology.

ABSTRACT
Copper, a strictly regulated trace element, is essential for many physiological processes including angiogenesis. Dysregulated angiogenesis has been associated with increased copper in tumors, and thus copper chelators have been used to inhibit tumor angiogenesis. However, it remains unclear whether copper has any effect on epithelial-mesenchymal transition (EMT). Using CoCl2-induced EMT of human breast carcinoma MCF-7 cells, we found that TEPA, a copper chelator, inhibited EMT-like cell morphology and cytoskeleton arrangement triggered by CoCl2; decreased the expression of vimentin and fibronectin, markers typical of EMT; inhibited HIF-1 activation and HIF1-α accumulation in nuclear; and down-regulated the expression of hypoxia-associated transcription factors, Snail and Twist1. Moreover, knockdown copper transport protein, Ctr1, also inhibited CoCl2-induced EMT and reversed the mesenchymal phenotype. In EMT6 xenograft mouse models, TEPA administration inhibited the tumor growth and increased mice survival. Immunohistochemical analysis of the xenograft further demonstrated that TEPA administration significantly inhibited tumor angiogenesis, down-regulated hypoxia-induced transcription factors, Snail and Twist1, leading to decreased transactivation of EMT-associated marker genes, vimentin and fibronectin. These results indicate that TEPA inhibits CoCl2-induced EMT most likely via HIF1-α-Snail/Twist signaling pathway, and copper depletion may be exploited as a therapeutic for breast cancer.

No MeSH data available.


Related in: MedlinePlus

TEPA affects the expression of typical EMT marker genes.(A) Expression of E-cadherin in MCF-7 maintained for 24 h under control, 200 μM CoCl2 conditions or treatment with 50 μM TEPA. Scale bars = 50 μm. (B) Expression of vimentin in MCF-7 maintained for 24 h under normoxic condition, hypoxia condition or treatment with TEPA. Scale bars = 50 μm.
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f5: TEPA affects the expression of typical EMT marker genes.(A) Expression of E-cadherin in MCF-7 maintained for 24 h under control, 200 μM CoCl2 conditions or treatment with 50 μM TEPA. Scale bars = 50 μm. (B) Expression of vimentin in MCF-7 maintained for 24 h under normoxic condition, hypoxia condition or treatment with TEPA. Scale bars = 50 μm.

Mentions: To assess effects of copper chelator on CoCl2-induced EMT of MCF-7 cells, TEPA, a specific copper chelator, was used to deplete the copper in culture medium. After 24-h treatment with TEPA (50 μM), few elongated and spindle-like mesenchyme-like cells were observed in either control or TEPA-treated groups (Fig. 3A). In contrast, cells under hypoxia-induced by CoCl2 (200 μM) exhibited epithelial cobblesone-like morphology (Fig. 3A). Similar results were obtained by quantitative evaluation of cell polarity. Copper depletion with TEPA showed 10% cells with polarity value above 2, which was similar to 6% cells with polarity value above 2 in control group (Fig. 3B). Cytoskeleton rearrangement and the formation of stress fibers were observed under oil objective (Fig. 3C). After 24 h treatment with CoCl2, cells exhibited typical fibroblast-like phenotype with actin filaments bundled into thick contractile stress fibers at the ventral cell surface, whereas cells treated with both CoCl2 and TEPA showed cortical thin bundles of actin filaments that were typical in control group. Furthermore, compared to cells treated with CoCl2, TEPA treatment reduced both nuclear (Fig. 4A,B) and total (Fig. 4D,E) accumulation of HIF1-α; significantly increased the expression of E-cadherin, an epithelial marker (Fig. 4D,E); and significantly decreased the expression of vimentin and fibronectin, markers of mesenchymal cells, at both mRNA and protein levels (Fig. 4C–E) as evaluated by both real-time PCR and Western blotting, respectively. Moreover, immunofluorescence data (Fig. 5) and quantitative analysis (Fig. S1) also demonstrated TEPA treatment increased the expression of E-cadherin and down-regulated expression of vimentin. Taken together, these results suggest that TEPA inhibited CoCl2-induced EMT of MCF-7 cells.


Copper depletion inhibits CoCl2-induced aggressive phenotype of MCF-7 cells via downregulation of HIF-1 and inhibition of Snail/Twist-mediated epithelial-mesenchymal transition.

Li S, Zhang J, Yang H, Wu C, Dang X, Liu Y - Sci Rep (2015)

TEPA affects the expression of typical EMT marker genes.(A) Expression of E-cadherin in MCF-7 maintained for 24 h under control, 200 μM CoCl2 conditions or treatment with 50 μM TEPA. Scale bars = 50 μm. (B) Expression of vimentin in MCF-7 maintained for 24 h under normoxic condition, hypoxia condition or treatment with TEPA. Scale bars = 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4502431&req=5

f5: TEPA affects the expression of typical EMT marker genes.(A) Expression of E-cadherin in MCF-7 maintained for 24 h under control, 200 μM CoCl2 conditions or treatment with 50 μM TEPA. Scale bars = 50 μm. (B) Expression of vimentin in MCF-7 maintained for 24 h under normoxic condition, hypoxia condition or treatment with TEPA. Scale bars = 50 μm.
Mentions: To assess effects of copper chelator on CoCl2-induced EMT of MCF-7 cells, TEPA, a specific copper chelator, was used to deplete the copper in culture medium. After 24-h treatment with TEPA (50 μM), few elongated and spindle-like mesenchyme-like cells were observed in either control or TEPA-treated groups (Fig. 3A). In contrast, cells under hypoxia-induced by CoCl2 (200 μM) exhibited epithelial cobblesone-like morphology (Fig. 3A). Similar results were obtained by quantitative evaluation of cell polarity. Copper depletion with TEPA showed 10% cells with polarity value above 2, which was similar to 6% cells with polarity value above 2 in control group (Fig. 3B). Cytoskeleton rearrangement and the formation of stress fibers were observed under oil objective (Fig. 3C). After 24 h treatment with CoCl2, cells exhibited typical fibroblast-like phenotype with actin filaments bundled into thick contractile stress fibers at the ventral cell surface, whereas cells treated with both CoCl2 and TEPA showed cortical thin bundles of actin filaments that were typical in control group. Furthermore, compared to cells treated with CoCl2, TEPA treatment reduced both nuclear (Fig. 4A,B) and total (Fig. 4D,E) accumulation of HIF1-α; significantly increased the expression of E-cadherin, an epithelial marker (Fig. 4D,E); and significantly decreased the expression of vimentin and fibronectin, markers of mesenchymal cells, at both mRNA and protein levels (Fig. 4C–E) as evaluated by both real-time PCR and Western blotting, respectively. Moreover, immunofluorescence data (Fig. 5) and quantitative analysis (Fig. S1) also demonstrated TEPA treatment increased the expression of E-cadherin and down-regulated expression of vimentin. Taken together, these results suggest that TEPA inhibited CoCl2-induced EMT of MCF-7 cells.

Bottom Line: Using CoCl2-induced EMT of human breast carcinoma MCF-7 cells, we found that TEPA, a copper chelator, inhibited EMT-like cell morphology and cytoskeleton arrangement triggered by CoCl2; decreased the expression of vimentin and fibronectin, markers typical of EMT; inhibited HIF-1 activation and HIF1-α accumulation in nuclear; and down-regulated the expression of hypoxia-associated transcription factors, Snail and Twist1.Immunohistochemical analysis of the xenograft further demonstrated that TEPA administration significantly inhibited tumor angiogenesis, down-regulated hypoxia-induced transcription factors, Snail and Twist1, leading to decreased transactivation of EMT-associated marker genes, vimentin and fibronectin.These results indicate that TEPA inhibits CoCl2-induced EMT most likely via HIF1-α-Snail/Twist signaling pathway, and copper depletion may be exploited as a therapeutic for breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Biophysics, School of Life Science and Technology.

ABSTRACT
Copper, a strictly regulated trace element, is essential for many physiological processes including angiogenesis. Dysregulated angiogenesis has been associated with increased copper in tumors, and thus copper chelators have been used to inhibit tumor angiogenesis. However, it remains unclear whether copper has any effect on epithelial-mesenchymal transition (EMT). Using CoCl2-induced EMT of human breast carcinoma MCF-7 cells, we found that TEPA, a copper chelator, inhibited EMT-like cell morphology and cytoskeleton arrangement triggered by CoCl2; decreased the expression of vimentin and fibronectin, markers typical of EMT; inhibited HIF-1 activation and HIF1-α accumulation in nuclear; and down-regulated the expression of hypoxia-associated transcription factors, Snail and Twist1. Moreover, knockdown copper transport protein, Ctr1, also inhibited CoCl2-induced EMT and reversed the mesenchymal phenotype. In EMT6 xenograft mouse models, TEPA administration inhibited the tumor growth and increased mice survival. Immunohistochemical analysis of the xenograft further demonstrated that TEPA administration significantly inhibited tumor angiogenesis, down-regulated hypoxia-induced transcription factors, Snail and Twist1, leading to decreased transactivation of EMT-associated marker genes, vimentin and fibronectin. These results indicate that TEPA inhibits CoCl2-induced EMT most likely via HIF1-α-Snail/Twist signaling pathway, and copper depletion may be exploited as a therapeutic for breast cancer.

No MeSH data available.


Related in: MedlinePlus