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. 2011;6(6):e20858.
doi: 10.1371/journal.pone.0020858. Epub 2011 Jun 8.

Role and mechanism of arsenic in regulating angiogenesis

Ling-Zhi Liu  1 , Yue Jiang, Richard L Carpenter, Yi Jing, Stephen C Peiper, Bing-Hua Jiang
Affiliations

Role and mechanism of arsenic in regulating angiogenesis

Ling-Zhi Liu et al. PLoS One. 2011.

Abstract

Arsenic is a wide spread carcinogen associated with several kinds of cancers including skin, lung, bladder, and liver cancers. Lung is one of the major targets of arsenic exposure. Angiogenesis is the pivotal process during carcinogenesis and chronic pulmonary diseases, but the role and mechanism of arsenic in regulating angiogenesis remain to be elucidated. In this study we show that short time exposure of arsenic induces angiogenesis in both human immortalized lung epithelial cells BEAS-2B and adenocarcinoma cells A549. To study the molecular mechanism of arsenic-inducing angiogenesis, we find that arsenic induces reactive oxygen species (ROS) generation, which activates AKT and ERK1/2 signaling pathways and increases the expression of hypoxia-inducible factor 1 (HIF-1) and vascular endothelial growth factor (VEGF). Inhibition of ROS production suppresses angiogenesis by decreasing AKT and ERK activation and HIF-1 expression. Inhibition of ROS, AKT and ERK1/2 signaling pathways is sufficient to attenuate arsenic-inducing angiogenesis. HIF-1 and VEGF are downstream effectors of AKT and ERK1/2 that are required for arsenic-inducing angiogenesis. These results shed light on the mechanism of arsenic in regulating angiogenesis, and are helpful to develop mechanism-based intervention to prevent arsenic-induced carcinogenesis and angiogenesis in the future.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Arsenic induced angiogenesis.
(A) A549 cells were treated without or with 5 µM of arsenic (As) for 5 h, then trypsinized, resuspended in serum-free medium (3×107cells/ml, 0.1 ml), and mixed in 1∶1 ratio with Matrigel (Collaborative Biomedical Products, Bedford, MA). Aliquots of the mixture were then implanted onto the CAM of 9-day-old embryos. After 96 h incubation, the area around the implanted Matrigel was photographed with a Nikon digital camera. Bar: 2 mm (upper panel). The number of blood vessels was obtained by counting the branching of blood vessels, and the relative angiogenesis was obtained by normalizing to that of the control without arsenic treatment. The data represent the mean ± SD of the relative angiogenesis from eight different embryos (bottom panel). *, indicates that the relative angiogenesis index significantly increased in arsenic treatment group when compared with control group, P<0.05. (B) BEAS-2B cells were treated with or without arsenic to perform tumor angiogenesis assay as above. Bar: 2 mm. *, indicates that the relative angiogenesis index significantly increased in arsenic treatment group when compared with control group, P<0.05.
Figure 2
Figure 2. Arsenic treatment induced phospho-AKT and phospho-ERK1/2 activation, and increased HIF-1α and VEGF expression.
(A) A549 and BEAS-2B cells were treated with different doses of arsenic (As) for 6 h, total proteins are subjected to Western blotting for HIF-1α and HIF-1β expression. A549 and BEAS-2B cells were cultured in serum-free medium for 24 h, then treated with different doses of arsenic for 2 h. Total proteins were subjected to Western blotting analysis for the levels of phospho-AKT, total AKT, phospho-ERK1/2, and ERK2 expression (upper panel). Relative densities of p-AKT, p-ERK1/2 and HIF-1α were analyzed by the ratio of p-AKT/AKT, p-ERK1/2/ERK2 and HIF-1α/HIF-1β using ImageJ software and normalized to those of control cells. The data represents the mean± SD from duplicate experiments (bottom panel). *, indicates significant increase when compared with the control cells, P<0.05. (B) BEAS-2B cells were seeded in 12-well plate. Cells were co-transfected with VEGF reporter and β-galactosidase (β-gal) plasmids and cultured for 15 h. Arsenic at 0, 2.5, and 5 µM was added for 24 h. Luciferase assay was performed by using luciferase assay system. The activity of β-gal was used as internal control of transfection efficiency. The relative luciferase activity was calculated as the ratio of luciferase/β-gal activity, and normalized to the control group. *, indicates that the relative luc activity significantly increased in arsenic treatment group when compared with the control group, P<0.05. C, BEAS-2B cells were treated without or with 5 µM of arsenic for 24 h. Total RNAs were extracted by Trizol and subjected to RT-PCR analysis of VEGF and GAPDH expression.
Figure 3
Figure 3. AKT and ERK1/2 pathways are required for arsenic-inducing HIF-1α expression and angiogenesis.
(A) BEAS-2B cells were cultured in serum-free medium for 24 h, then cells were pre-treated with LY294002 or U0126 at 20 µM for 30 min. Arsenic at 5 µM was added to the cells for 2 h (for p-AKT, AKT, p-ERK1/2, and ERK2 expression) and 6 h (for HIF-1α and HIF-1β expression), respectively. Total proteins were analyzed by Western blotting to detect the expression of proteins as indicated (left panel). Relative densities of p-AKT, p-ERK1/2 and HIF-1α were analyzed as Fig. 2A (right panel). *, indicates significant increase when compared with the control cells, P<0.05. . #, indicates significant decrease when compared with the sodium arsenite treatment, P<0.05. (B) BEAS-2B cells were treated with 5 µM of arsenic. Cells were trypsinized, mixed with equal volume of Matrigel with or without 15 µM of LY294002 or U0126. Equal volume of DMSO was added as a negative control. Angiogenesis assay was performed as described in Fig. 1. *, indicates that the relative angiogenesis index was significantly decreased when compared with DMSO control group, P<0.05. (C) BEAS-2B cells were infected with adenovirus carrying GFP (Ad-GFP) or AKT dominant negative (Ad-AKT-DN) at 20 MOI (upper panel), or transfected with scrambled control of siRNA (siScramble) or siMAPK at 50 nM (bottom panel). After 24 h, cells were treated with arsenic and angiogenesis assay was performed as above. *, indicates that the relative angiogenesis index was significantly decreased when compared with Ad-GFP or siScramble control group, P<0.05.
Figure 4
Figure 4. Arsenic induced ROS production in BEAS-2B cells, which was required for angiogenesis.
(A) BEAS-2B cells were seeded into 6-well plates. Cells were treated with different doses of arsenic as indicated in serum-free medium. DCFH-DA at 5 µM was added to the cells for 15 min. Then the cells were washed and fixed, and the fluorescent images were captured using a fluorescent microscope (upper panel). The corresponding phase micrographs were shown in the bottom panel. (B) BEAS-2B cells were seeded into the 6-well plate. The cells were then cultured in serum-free medium with arsenic at 5 µM for different time points as indicated. DCFH-DA staining was performed as above. (C) BEAS-2B cells were infected with adenovirus carrying GFP (Ad-GFP) and catalase (Ad-catalase), respectively at 20 MOI. After 24 h, cells were treated with 5 µM arsenic for 5 h to perform angiogenesis assay. *, indicates that the relative angiogenesis index was significantly decreased when compared with Ad-GFP control group, P<0.05.
Figure 5
Figure 5. ROS are required for AKT and ERK1/2 activation, HIF-1α expression, and angiogenesis.
(A) BEAS-2B cells were cultured in serum-free medium for 24 h, then cells were pre-treated with DPI or catalase for 30 min. Arsenic at 5 µM was added to the cells for 2 h or 6 h as above. The specific proteins are analyzed by Western blotting (left panel). The relative densities of p-AKT, p-ERK1/2 and HIF-1α were determined as above. *, indicates significant increase when compared with the control cells, P<0.05. . #, indicates significant decrease when compared with the sodium arsenite treatment alone, P<0.05. (B) BEAS-2B cells were infected with adenovirus carrying GFP and HIF-1α siRNA (Ad-GFP and Ad-siHIF-1α, respectively) at 20 MOI for 24 h, then the cells were treated with 5 µM arsenic for 5 h and angiogenesis assay was performed as above. *, indicates that the relative angiogenesis index was significantly decreased when compared with the control group, P<0.05.
Figure 6
Figure 6. VEGF is required for arsenic-inducing angiogenesis.
(A) BEAS-2B cells were treated with DPI or catalase for 30 min, then with 5 µM arsenic for 24 h. Total RNAs were extracted by Trizol, and analyzed by RT-PCR for VEGF and GAPDH expression (upper panel). Relative density of VEGF was analyzed by the ratio of VEGF/GAPDH using ImageJ software and normalized to control cells. The data represents the mean± SD from duplicate experiments (bottom panel). *, indicates significant increase when compared with the control cells, P<0.05. . #, indicates significant decrease when compared with the sodium arsenite treatment alone, P<0.05. (B) BEAS-2B cells were transfected with VEGF siRNA and scrambled siRNA (siVEGF and siScramble, respectively). After the transfection for 24 h, cells were treated with 5 µM arsenic for 5 h to perform angiogenesis assay. *, indicates that the relative angiogenesis index was significantly decreased in siVEGF treatment group when compared with siScramble group, P<0.05.
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