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. 2013 Aug 15;271(1):20-9.
doi: 10.1016/j.taap.2013.04.018. Epub 2013 Apr 30.

Epithelial to mesenchymal transition in arsenic-transformed cells promotes angiogenesis through activating β-catenin-vascular endothelial growth factor pathway

Zhishan Wang  1 , Brock Humphries, Hua Xiao, Yiguo Jiang, Chengfeng Yang
Affiliations

Epithelial to mesenchymal transition in arsenic-transformed cells promotes angiogenesis through activating β-catenin-vascular endothelial growth factor pathway

Zhishan Wang et al. Toxicol Appl Pharmacol. .

Abstract

Arsenic exposure represents a major health concern increasing cancer risks, yet the mechanism of arsenic carcinogenesis has not been elucidated. We and others recently reported that cell malignant transformation by arsenic is accompanied by epithelial to mesenchymal transition (EMT). However, the role of EMT in arsenic carcinogenesis is not well understood. Although previous studies showed that short term exposure of endothelial cells to arsenic stimulated angiogenesis, it remains to be determined whether cells that were malignantly transformed by long term arsenic exposure have a pro-angiogenic effect. The objective of this study was to investigate the effect of arsenic-transformed human bronchial epithelial cells that underwent EMT on angiogenesis and the underlying mechanism. It was found that the conditioned medium from arsenic-transformed cells strongly stimulated tube formation by human umbilical vein endothelial cells (HUVECs). Moreover, enhanced angiogenesis was detected in mouse xenograft tumor tissues resulting from inoculation of arsenic-transformed cells. Mechanistic studies revealed that β-catenin was activated in arsenic-transformed cells up-regulating its target gene expression including angiogenic-stimulating vascular endothelial growth factor (VEGF). Stably expressing microRNA-200b in arsenic-transformed cells that reversed EMT inhibited β-catenin activation, decreased VEGF expression and reduced tube formation by HUVECs. SiRNA knockdown β-catenin decreased VEGF expression. Adding a VEGF neutralizing antibody into the conditioned medium from arsenic-transformed cells impaired tube formation by HUVECs. Reverse transcriptase-PCR analysis revealed that the mRNA levels of canonical Wnt ligands were not increased in arsenic-transformed cells. These findings suggest that EMT in arsenic-transformed cells promotes angiogenesis through activating β-catenin-VEGF pathway.

Keywords: Angiogenesis; Arsenic-transformed cells; EMT; Epithelial-to-mesenchymal transition (EMT); HUVEC; IF; MicroRNA-200b (miR-200b); VEGF; Wnt; epithelial-to-mesenchymal transition; human bronchial epithelial cells (HBECs) with p53 expression stably knocked down; human umbilical vein endothelial cell; immunofluorescence; miR-200b; microRNA 200b; p53(low)HBECs; siRNA; small interfering RNA; vascular endothelial growth factor; β-Catenin.

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Figures

Fig. 1
Fig. 1. Epithelial to mesenchymal transition (EMT) in arsenic-transformed cells (As-p53lowHBECs) promotes angiogenesis
(A) The conditioned medium from arsenic-transformed cells stimulates tube formation by HUVECs and (B) Stably expressing miR-200b in arsenic-transformed cells impairs tube formation by HUVECs. Representative images of tube formation by HUVECs induced by the conditioned media from indicated cells. The conditioned media were prepared for tube formation assay as described in Materials and Methods. The quantifications of formed tube branches was carried out as described in Materials and Methods and presented as total number of tube branches per well (means ± standard deviations, n=3). Scale bar=200 μm. * p<0.05, compared to p53lowHBECs (A) and to As-p53lowHBEC-GFP (B). Similar results were obtained in two additional experiments. (C) Enhanced angiogenesis is detected in mouse xenograft tumors produced by inoculation of arsenic-transformed cells (As-p53lowHBEC-GFP).Representative overlaid fluorescent images from anti-CD31 immunofluorescence staining (red color) and nucleus DAPI staining (blue color) in mouse xenograft tissues resulting from injection of As-p53lowHBEC-GFP or As-p53lowHBEC-GFP-200b cells. Tissue section preparation and anti-CD31 staining were carried out as described in Materials and Methods. The CD31 staining was quantified and presented as the number of CD31 positive-stained vessel structures per field of view (FOV) as described in Materials and Methods (means ± standard deviations, n=3). Scale bar=100 μm. * p<0.05, compared to the As-p53lowHBEC-GFP group.
Fig. 2
Fig. 2. β-Catenin is activated in arsenic-transformed human bronchial epithelial cells (As-p53lowHBECs)
(A) Representative images of control untransformed p53lowHBECs shown in a bright field (upper panel) and their β-catenin immunofluorescence staining (lower pane). White arrows point to representative cytoplasmic membrane stainings of β-catenin. (B) Representative images of arsenic-transformed cells (As-p53lowHBECs) shown in a bright field (upper panel) and their β-catenin immunofluorescence staining (lower pane). White arrows point to representative nucleus stainings of β-catenin. β-Catenin immunofluorescence staining was carried out as described in Materials and Methods. Scale bar=50 μm. (C) Representative Western blot analysis of cellular β-catenin, c-myc, cyclin D1 and VEGF protein levels. Western blot was carried out as described in Materials and Methods. (D) Quantification of cellular TOPflash and FOPflash reporter luciferase activity. The TOPflash and FOPflash reporter luciferase activity was measured using a dual luciferase reporter assay and calculated as described in Materials and Methods. The results are expressed as the ratio of the TOPflash or FOPflash luciferase activity divided by the Renilla luciferase activity (means ± standard deviations, n=3). * p<0.05, compared to p53lowHBECs. Similar results were obtained in two additional experiments.
Fig. 3
Fig. 3. Stably re-expressing miR-200b in arsenic-transformed cells restores β-catenin cytoplasmic membrane localization and reduces its target gene expression
(A) Representative images of arsenic-transformed vector control cells (As-p53lowHBEC-GFP) shown in a bright field (upper panel) and their β-catenin immunofluorescence staining (lower pane). White arrows point to representative nucleus stainings of β-catenin. (B) Representative images of arsenic-transformed miR-200b stably expressing cells (As-p53lowHBEC-GFP-200b) shown in a bright field (upper panel) and their β-catenin immunofluorescence staining (lower pane). White arrows point to representative cytoplasmic membrane stainings of β-catenin. β-Catenin immunofluorescence staining was carried out as described in Materials and Methods. Scale bar=50 μm. (C) Representative Western blot analysis of cellular β-catenin, c-myc, cyclin D1 and VEGF protein levels. Western blot was carried out as described in Materials and Methods. (D) Q-PCR analysis of cellular c-myc, cyclin D1 and VEGF mRNA levels (means ± standard deviations, n=3). Q-PCR analysis was performed as described in Materials and Methods. * p<0.05, compared to the As-p53lowHBEC-GFP cells. Similar results were obtained in two additional experiments.
Fig. 4
Fig. 4. SiRNA knocking down β-catenin expression in arsenic-transformed cells reduces VEGF expression and impairs their conditioned medium-induced tube formation by HUVECs
(A, B) Representative images of tube formation by HUVECs (A) and quantification of formed tube branches (means ± standard deviations, n=3) (B) induced by the conditioned medium from As-p53lowHBECs transfected with Control or β-catenin siRNA. Scale bar=200 μm. * p<0.05, compared to Control siRNA-transfected cells. (C) Representative Western blot analysis of cellular β-catenin, c-myc, cyclin D1 and VEGF protein levels. (D) Conditioned medium VEGF levels measured by ELISA (means ± standard deviations, n=3). * p<0.05, compared to Control siRNA-transfected cells. After overnight culture, As-p53lowHBECs were transfected with negative Control or β-catenin siRNA oligoes as described in Materials and Methods. Conditioned medium were collected for tube formation assay and ELISA measurement of VEGF levels and Western blot was performed as described in Materials and Methods. Similar results were obtained in two additional experiments.
Fig. 5
Fig. 5. Adding a VEGF neutralizing antibody into the conditioned medium from arsenic-transformed cells reduces tube formation by HUVECs
(A, B) Representative images of tube formation by HUVECs (A) and quantification of formed tube branches (means ± standard deviations, n=3) (B) induced by the conditioned medium from As-p53lowHBECs with the addition of a Control antibody or a VEGF neutralizing antibody. Scale bar=200 μm. * p<0.05, compared to Control antibody (Ab) group. Conditioned medium were collected for tube formation assay as described in Materials and Methods. Similar results were obtained in two additional experiments.
Fig. 6
Fig. 6. RT-PCR analysis of cellular Wnt ligands and their Frizzled receptors (FZDs)
(A, B) Representative images of RT-PCR analysis of cellular Wnt ligands (A) and their Frizzled receptors (B). About 80-90% confluence of control untransformed cells (p53lowHBECs), arsenic-transformed cells (As-p53lowHBECs), vector control (As-p53lowHBEC-GFP) or miR-200b stably expressing (As-p53lowHBEC-200b) cells were used for extracting total RNA for RT-PCR analysis as described in Materials and Methods. Similar results were obtained in two additional experiments.
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