Role and Mechanism of Arsenic in Regulating Angiogenesis

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.

About the Authors:

Ling-Zhi Liu

Contributed equally to this work with: Ling-Zhi Liu, Yue Jiang

Affiliation: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America

Yue Jiang

Contributed equally to this work with: Ling-Zhi Liu, Yue Jiang

Affiliation: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America

Richard L. Carpenter

Affiliation: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America

Yi Jing

Affiliation: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America

Stephen C. Peiper

Affiliation: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America

Bing-Hua Jiang

* E-mail: [email protected]

Affiliation: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America

Introduction

Arsenic is a well-documented carcinogen associated with human lung, skin, urinary bladder, kidney and liver cancers [1]–[3]. Arsenic causes DNA-strand break, loss of DNA methylation, cellular endocrine disorder and cell transformation [4]–[9]. Arsenic also induces oxidative stress, genotoxic damage, DNA repair inhibition, epigenetic events and activation of certain signal transduction pathways leading to aberrant gene expression [10]. The WHO sets the maximum safe level of arsenic at 0.01 mg/L, while arsenic levels in water in some areas can be up to several thousand times higher than the limit [11]. The lung cancer is highly associated with arsenic exposure [1], [12], [13]. In addition to lung cancer, arsenic exposure also leads to other cancers and non-malignant pulmonary diseases including chronic bronchitis and chronic obstructive pulmonary diseases (COPD) [14]. However, the mechanisms of arsenic exposure leading to the lung carcinogenesis and other pulmonary diseases remain to be elucidated.

Angiogenesis is the process that new capillaries are generated from the pre-existing vasculature which plays a pivotal role in the initiation of carcinogenesis and tumor progress, vascular diseases, and various ischemic and inflammatory diseases [15], [16]. Tumor cannot grow without angiogenesis when it reaches 1∼2 mm in diameter [17]. Angiogenesis is also a prominent feature of the structural tissue remodeling that occurs in the chronic airway diseases including COPD, in which the angiogenesis/angiostatic balance is affected [18], [19]. VEGF is the most potent angiogenic factor. Our previous studies demonstrated that arsenic induced VEGF expression in human prostate cancer cells and tumor angiogenesis [20], [21]. Acute arsenic treatment also induced VEGF expression in human microvascular endothelial cells through the induction of heme oxygenase-1 gene expression [22]. It has been reported that sodium arsenite (arsenic) treatment induced angiogenesis responses using the chick chorioallantoic membrane (CAM) model [23]. Recent study showed that the chronic exposure of arsenic in drinking water caused both liver angiogenesis and pathogenic liver sinusoidal endothelial cell capillarization in mice mediated by sphingosine-1-phosphate type 1 receptor in endothelial cells [24]. However, it is unknown whether acute arsenic exposure may affect epithelial cells to induce angiogenesis in vivo.

In this study, we used human immortalized normal lung epithelial cells BEAS-2B to determine: 1) whether short time exposure of arsenic induces angiogenesis; 2) what signaling pathways are involved in arsenic-inducing angiogenesis; 3) whether VEGF and HIF-1 are required for arsenic-inducing angiogenesis. Given the important role of angiogenesis in lung cancer and other arsenic-induced pulmonary diseases, these results may shed light on the mechanism of arsenic-induced angiogenesis and help to develop mechanism-based interventions to prevent carcinogenesis and other diseases induced by arsenic in the future.

Materials and Methods

Cell Culture

Human immortalized lung epithelial cells, BEAS-2B cells (ATCC, Manassas, VA) were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 100 units/ml penicillin, and 100 µg/ml streptomycin, and cultured at 37°C in a 5% CO₂ incubator. Human lung carcinoma cells, A549 cells (ATCC, Manassas, VA) were maintained in RPMI 1640 medium with 10% FBS and antibiotics as above.

Angiogenesis Assay

Fertilized chicken eggs (Charles River Laboratories International, Inc., Wilmington, MA) were incubated at 37°C for 9 days, and angiogenesis assay was performed as we previously described [25], [26]. In brief, BEAS-2B cells and A549 cells were treated with 5 µM of sodium arsenic (Sigma, St. Louis, MO) for 5 h. Then cells were trypsinized, resuspended, mixed with Matrigel, without or with LY294002 (PI3K inhibitor) and U0126 (MAPK/ERK inhibitor), and implanted onto the CAM at Day 9. Adenoviruses carrying GFP, AKT-DN, HIF-1α siRNA (si HIF-1α), and catalase were used to infect BEAS-2B cells at 30 MOI for 24 h before the arsenic treatment. Similarly, BEAS-2B cells were transfected by siRNA oligomers of siScrambled control (siSCR), MAPK siRNA (siMAPK), and VEGF (siVEGF) at 50 nM for 24 h, then treated by 5 µM sodium arsenite for 5 h. The siRNA against MAPK was purchased from Qiagen. The sense sequence: UGCUGACUCCAAAGCUCUGdTdT ; the antisense sequence: CAGAGCUUUGGAGUCAGCAdTdT . The On-Target plus Smartpool siVEGF was from Dharmacon (Thermo Scientific, Pittsburgh, PA). The four target sequences are: GCAGAAUCAUCACGAAGUG; CAACAAAUGUGAAUGCAGA; GGAGUACCCUGAUGAGAUC; GAUCAAACCUCACCAAGGC. The cells were used for angiogenesis assay. After 96 h of incubation, the tumor plugs were trimmed off the CAM, and analyzed for angiogenesis response. The number of blood vessels as the index of angiogenesis was obtained by counting the branching of blood vessels in 2 square millimeter, which was normalized to that of the control group.

Immunoblotting

BEAS-2B cells were starved for 24 h, then treated with sodium arsenite for 2 h (for p-AKT, AKT, p-ERK1/2 and ERK2 expression) or 6 h (for HIF-1α and HIF-1β expression). To study the effects of inhibitors, starved BEAS-2B cells were pre-treated with diphenylene iodonium (DPI), catalase, LY294002, or U0126 for 30 min, then treated with 5 µM of sodium arsenite as above. Total proteins were extracted from the cells using modified RIPA buffer supplemented with protein inhibitors, separated by SDS-PAGE, and blotted onto nitrocellular membrane. After blocking with 5% nonfat milk for 2 h, the membranes were incubated with the primary antibodies against HIF-1α, HIF-1β (BD, Franklin Lakes, NJ), phospho-AKT, phospho-ERK1/2, AKT (Cell Signaling Technology, Beverly, MA ), or ERK2 (Santa Cruz Biotechnology, Santa Cruz, CA) overnight. The specific protein signals were detected by incubation with horseradish peroxidase-conjugated antibodies and with a chemiluninescence reagent (Pierce, Rockford, IL) [27].

Dichlorofluorescein diacetate (DCFH-DA) Staining for ROS

Cells were treated with sodium arsenite at different concentrations for 2 h, or treated with 5 µM sodium arsenite for different time points as indicated, then cells were incubated with DCFH- DA dye (Molecular Probe, Eugene, OR) for 15 min, washed with PBS buffer, and fixed with10% formaldehyde solution. The images were taken using a fluorescent microscope and analyzed as we previously described [26], [27].

Luciferase Assay

Cells were seeded in 12-well plate at 60% confluence and cultured overnight. Cells were co-transfected with VEGF reporter and β-galactosidase (β-gal) plasmids and cultured for 15 h. Sodium arsenite at 0, 2.5, and 5 µM was added to the cells for 24 h. Luciferase assay was analyzed using luciferase assay system (Promega, San Luis Obispo, CA). The activity of β-gal was used as internal control of transfection efficiency. The relative luciferase (luc) activity was calculated as the ratio of luciferase/β-gal, and normalized to the control group [27].

RT-PCR

Total RNAs were extracted using Trizol reagent (Invitrogen, Carlsbad, CA). Primers used for detecting VEGF and GAPDH are as follows:

VEGF forward primer, 5′-TCGGGCCTCCGAAACCATGA-3′;

VEGF reverse primer, 5′-CCTGGTGAGAGATCTGGTTC-3′;

GAPDH forward primer, 5′- CCACCCATGGCAAATTCCATGGCA-3′;

GAPDH reverse primer, 5′-TCTAGACGGCAGGTCAGGTCCACC-3′.

The amplification of VEGF was performed by PCR as previously described [27]. The expression levels of GAPDH were used as an internal control.

Statistical Analysis

All values in this study were reported as mean ± SD. Student's unpaired t test was used for statistical analyses. The values were considered as significant difference at p<0.05.

Results

Arsenic treatment induces angiogenesis

Angiogenesis is a vital pathological process during tumorigenesis. To investigate whether short time treatment of arsenic has effect on angiogenesis, human lung adenocarcinoma cell line A549 and immortalized normal human lung epithelial cells BEAS-2B were treated with 5 µM of sodium arsenite for 5 h. Angiogenesis assay was performed using the cells on the CAM model. Arsenic treatment strongly increased A549 cell-inducing angiogenesis to 2.5-fold when compared to the untreated control cells (Fig. 1A). Similarly, arsenic treatment also promoted BEAS-2B cells to induce angiogenesis by more than 2-fold (Fig. 1B). These results suggest that arsenic treatment induces angiogenesis in both immortalized lung epithelial cells and lung cancer cells.

[Figure omitted. See PDF.]

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.

https://doi.org/10.1371/journal.pone.0020858.g001

Arsenic activates AKT and ERK1/2 signaling pathways, and increases the expression of HIF-1α and VEGF

AKT (also known as Protein kinase B) is a serine/threonine kinase. The other important parallel signaling pathway is extracellular signal-regulated kinase (ERK). We showed that arsenic at 5 µM increased AKT and ERK1/2 activation in both A549 and BEAS-2B cells (Fig. 2A), suggesting that arsenic can activate these two signaling pathways in lung epithelial and cancer cells. To analyze the downstream effector of AKT and ERK1/2, we tested whether the expression of HIF-1 was induced by arsenic. HIF-1 is a heterodimeric transcription factor that is composed of a constitutive-expressing HIF-1β subunit and an oxygen-sensitive HIF-1α subunit [28]. A549 cells and BEAS-2B cells were treated with different doses of arsenic. As shown in Fig. 2A, arsenic treatment increased HIF-1α expression levels in both A549 and BEAS-2B cells. VEGF is a potent angiogenesis inducer that is upregulated by HIF-1 through the binding of HIF-1 to its promoter [29]. Similarly, VEGF transcriptional activation was significantly increased by arsenic treatment (Fig. 2B), and VEGF mRNA level was induced by arsenic treatment (Fig. 2C), suggesting that arsenic induces VEGF expression at transcriptional level.

[Figure omitted. See PDF.]

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.

https://doi.org/10.1371/journal.pone.0020858.g002

AKT and ERK1/2 are required for arsenic-inducing angiogenesis

Pre-treatment of BEAS-2B cells with PI3K and MAP kinase inhibitors LY294002 and U0126 blocked the activation of phospho-AKT or phospho-ERK induced by arsenic, and decreased arsenic-inducing HIF-1α but not HIF-1β protein levels (Fig. 3A). This result suggests that both AKT and ERK1/2 are required for arsenic-induced HIF-1 expression. To test whether AKT and ERK are required for arsenic-inducing angiogenesis, BEAS-2B cells were treated by LY294002 or U0126, and used to do angiogenesis assay. We found that LY294002 or U0126 treatment inhibited angiogenesis by more than 50% when compared to the control (Fig. 3B). To further specifically inhibit AKT or ERK, the cells were infected with adenoviruses carrying GFP (Ad-GFP) and AKT dominant negative (Ad-AKT-DN) to specifically inhibit AKT activation; or transfected with scrambled control siRNA and siMAPK to knockdown MAPK expression. These cells were used to perform angiogenesis assay. Similar to the effect of LY294002 and U0126 treatment, BEAS-2B cells treated with Ad-AKT-DN or siMAPK showed significant inhibition of angiogenesis responses with the reduction to 50% and 70% as compared to those of the Ad-GFP or siScrambled control group, respectively (Fig. 3C). These results further confirm that AKT and ERK1/2 are required for arsenic-inducing angiogenesis.

[Figure omitted. See PDF.]

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.

https://doi.org/10.1371/journal.pone.0020858.g003

Arsenic induces reactive oxygen species (ROS) generation, which is necessary for arsenic-inducing angiogenesis

Previous study showed that low level of sodium arsenic at 100 nM exposure for long time renders human keratinocytes apoptotic resistant and induces ROS generation [30]. To study whether short time exposure of arsenic may induce ROS levels, BEAS-2B cells were treated with different doses of arsenic for 2 h, or treated with 5 µM arsenic for 2 h and 4 h. As shown in Fig. 4A, ROS production levels were higher with arsenic treatment at 10 µM than at 5 µM, and were higher with arsenic treatment at 5 µM for 4 h than for 2 h (Fig. 4B). These results demonstrate that ROS are induced by arsenic treatment.

[Figure omitted. See PDF.]

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.

https://doi.org/10.1371/journal.pone.0020858.g004

To further test whether arsenic-inducing ROS are required for angiogenesis, BEAS-2B cells were infected with adenovirus carrying catalase, a scavenger of hydrogen peroxide, or GFP (as control) at 20 MOI. These cells were treated by arsenic and used for angiogenesis assay as previously described. Overexpression of catalase in arsenic-treated BEAS-2B cells significantly decreased angiogenesis when compared to the control group (Fig. 4C). These results indicate that arsenic induces ROS production which is required for arsenic-inducing angiogenesis.

ROS induction is required for arsenic-induced AKT and ERK1/2 activation and HIF-1 expression; and HIF-1 elevation is required for arsenic-induced angiogenesis

To explore the molecular mechanism of ROS in mediating angiogenesis, we showed that ROS inhibitors DPI and catalase significantly suppressed AKT and ERK1/2 activation (Fig. 5A). Similarly, the expression of HIF-1α was decreased by ROS inhibitors (Fig. 5A). To determine whether HIF-1α elevation is required for arsenic-induced angiogenesis, BEAS-2B cells were transduced by adenovirus carrying siHIF-1α or GFP. These cells were treated by arsenic to perform angiogenesis assay as above. As shown in Fig. 5B, HIF-1α knockdown markedly inhibited the angiogenesis with the reduction of angiogenesis responses by 50%. These results suggest that arsenic induces HIF-1 expression through ROS generation, AKT and ERK1/2 activation, and HIF-1 elevation is required for arsenic-induced angiogenesis.

[Figure omitted. See PDF.]

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.

https://doi.org/10.1371/journal.pone.0020858.g005

ROS production is required for arsenic-induced VEGF expression, and VEGF is required for arsenic-induced angiogenesis

It is known that HIF-1 induces VEGF transcription by the binding of HIF-1 to VEGF promoter [29]. Both HIF-1 and VEGF play a pivotal role in regulating anigogenesis [17]. To determine whether ROS are required for arsenic-induced VEGF expression, the cells were pretreated with DPI and catalase, followed by the arsenic treatment. DPI and catalase suppressed arsenic-induced VEGF expression (Fig. 6A). To assess whether VEGF expression is required for arsenic-induced angiogenesis, the cells were transfected with VEGF siRNA or scrambled siRNA control, followed by the treatment of arsenic. These treated cells were used to perform angiogenesis assay. Similar to the results of HIF-1α knockdown, VEGF knockdown also decreased arsenic-induced angiogenesis by 50% (Fig. 6B), suggesting that arsenic can increase HIF-1 expression, which in turn regulates VEGF expression for inducing angiogenesis.

[Figure omitted. See PDF.]

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.

https://doi.org/10.1371/journal.pone.0020858.g006

Discussion

Environmental exposure to arsenic is commonly in the forms of either arsenite (As⁺³) or arsenate (As⁺⁵), which is widely present in environments such as water, air and soil. Arsenite is the predominant form of arsenic in contaminated water and is the form mainly attributed to the induction of human cancers, including lung, skin, bladder, and liver cancer. People are exposed to arsenic through the arsenic-containing drinking water, food, and air-dust. The occupational exposure to arsenic may be through the inhalation of arsenic dusts in the production and distribution process. There are more than 1.5 million industrial workers that are potentially exposed to arsenic and arsenic compounds during the product manufacturing and distribution according to the NIOSH estimate [31]. Lung is one of the major organs to arsenic exposure. As a metalloid carcinogen, arsenic is known to be associated with higher incidence of both lung cancer and non-malignant pulmonary diseases. In this study, we used sodium arsenic to treat both human lung adenocarcinoma cells A549 and immortalized human lung epithelial cells, BEAS-2B; and demonstrated that short time exposure of arsenic in lung epithelial cells induced angiogenesis, suggesting that arsenic-inducing angiogenesis is involved in arsenic-inducing lung carcinogenesis.

ROS include superoxide, hydrogen peroxide and hydroxyl radical. Previous studies demonstrated that arsenic induced ROS production and DNA damage [4], [32]-[34]. We found that arsenic induced ROS production in a dose-dependent manner, and that suppression of ROS generation by catalase inhibited arsenic-inducing angiogenesis, suggesting that ROS production is required for arsenic-inducing angiogenesis.

In this study, we showed that arsenic treatment led to the induction of VEGF and HIF-1 expression. HIF-1 composes of the heterodimers of HIF-1α and HIF-1β subunits, which regulates VEGF expression at transcriptional level. VEGF is a key angiogenesis factor for stimulating endothelial cell growth and angiogenesis [35], [36]. Inhibition of VEGF-inducing angiogenesis greatly decreased tumor growth in vivo [37]. In chronic pulmonary diseases such as COPD, pulmonary endothelial dysfunction and change of levels of VEGF and its receptor were found to be involved in the development of vascular disease [38], [39]. HIF-1 also regulates many other target genes that are involved in crucial aspects of cancer biology including angiogenesis [28]. Overexpression of HIF-1 is a hallmark of many kinds of solid tumors, and is also associated with skeletal muscle disease COPD [28], [40], [41]. Recent study showed that chronic exposure to low-dose As⁺³ stimulated tumor growth through induction of angiogenesis [42]. This study showed that the knockdown of HIF-1 and VEGF using siRNAs against HIF-1α or VEGF inhibited arsenic-inducing angiogenesis, demonstrating the important role of HIF-1α and VEGF in arsenic-inducing angiogenesis.

To further determine the signaling pathways in upregulating HIF-1 and VEGF expression, we found that AKT and ERK1/2 were activated by arsenic, and that inhibition of AKT or ERK1/2 by their chemical inhibitors, dominant negative molecule, or siRNAs blocked arsenic-inducing angiogenesis. These findings suggest that AKT and ERK pathways are involved in arsenic-stimulating angiogenesis in lung epithelial cells, which is consistent with previous study showing that arsenic induced ROS generation and ERK activation [4]. This study reveals the important biological function of the previous observation by our lab and others that showed AKT and ERK are upstream regulators of HIF-1 and VEGF [27], [43]–[45]. We further demonstrated that inhibition of ROS generation blocked HIF-1 and VEGF induction, AKT and ERK1/2 activation, and angiogenesis in response to arsenic treatment. This new result provides an important link from ROS, signaling molecules, to angiogenesis. In addition to acute treatment of arsenic, we found that chronic treatment with sodium arsenite had similar effects.

Taken together, this study demonstrated that acute treatment of arsenic in lung epithelial cells induced angiogenesis, arsenic increases ROS induction for inducing angiogenesis, and ROS transmits the angiogenesis signals to downstream targets AKT, ERK1/2, HIF-1, and VEGF. This study shows a novel molecular mechanism of arsenic in affecting human lung epithelial cells to induce angiogenesis. This finding may provide useful information for developing mechanism-based prevention and treatment of lung cancer induced by arsenic in the future.

Author Contributions

Conceived and designed the experiments: BHJ LZL. Performed the experiments: LZL Yue Jiang RLC Yi Jing. Analyzed the data: Yue Jiang LZL RLC. Contributed reagents/materials/analysis tools: SCP. Wrote the paper: LZL SCP BHJ.

Citation: Liu L-Z, Jiang Y, Carpenter RL, Jing Y, Peiper SC, Jiang B-H (2011) Role and Mechanism of Arsenic in Regulating Angiogenesis. PLoS ONE 6(6): e20858. https://doi.org/10.1371/journal.pone.0020858

References

1. Brown LM, Pottern LM, Blot WJ (1984) Lung cancer in relation to environmental pollutants emitted from industrial sources. Environ Res 34: 250–261.LM BrownLM PotternWJ Blot1984Lung cancer in relation to environmental pollutants emitted from industrial sources.Environ Res34250261

2. Knobeloch LM, Zierold KM, Anderson HA (2006) Association of arsenic-contaminated drinking-water with prevalence of skin cancer in Wisconsin's Fox River Valley. J Health Popul Nutr 24: 206–213.LM KnobelochKM ZieroldHA Anderson2006Association of arsenic-contaminated drinking-water with prevalence of skin cancer in Wisconsin's Fox River Valley.J Health Popul Nutr24206213

3. Waalkes MP, Ward JM, Diwan BA (2004) Induction of tumors of the liver, lung, ovary and adrenal in adult mice after brief maternal gestational exposure to inorganic arsenic: promotional effects of postnatal phorbol ester exposure on hepatic and pulmonary, but not dermal cancers. Carcinogenesis 25: 133–141.MP WaalkesJM WardBA Diwan2004Induction of tumors of the liver, lung, ovary and adrenal in adult mice after brief maternal gestational exposure to inorganic arsenic: promotional effects of postnatal phorbol ester exposure on hepatic and pulmonary, but not dermal cancers.Carcinogenesis25133141

4. Chowdhury R, Chatterjee R, Giri AK, Mandal C, Chaudhuri K (2010) Arsenic-induced cell proliferation is associated with enhanced ROS generation, Erk signaling and CyclinA expression. Toxicol Lett 198: 263–271.R. ChowdhuryR. ChatterjeeAK GiriC. MandalK. Chaudhuri2010Arsenic-induced cell proliferation is associated with enhanced ROS generation, Erk signaling and CyclinA expression.Toxicol Lett198263271

5. Davey JC, Bodwell JE, Gosse JA, Hamilton JW (2007) Arsenic as an endocrine disruptor: effects of arsenic on estrogen receptor-mediated gene expression in vivo and in cell culture. Toxicol Sci 98: 75–86.JC DaveyJE BodwellJA GosseJW Hamilton2007Arsenic as an endocrine disruptor: effects of arsenic on estrogen receptor-mediated gene expression in vivo and in cell culture.Toxicol Sci987586

6. Dong JT, Luo XM (1993) Arsenic-induced DNA-strand breaks associated with DNA-protein crosslinks in human fetal lung fibroblasts. Mutat Res 302: 97–102.JT DongXM Luo1993Arsenic-induced DNA-strand breaks associated with DNA-protein crosslinks in human fetal lung fibroblasts.Mutat Res30297102

7. Reichard JF, Schnekenburger M, Puga A (2007) A Long term low-dose arsenic exposure induces loss of DNA methylation. Biochem Biophys Res Commun 352: 188–192.JF ReichardM. SchnekenburgerA. Puga2007A Long term low-dose arsenic exposure induces loss of DNA methylation.Biochem Biophys Res Commun352188192

8. Shi H, Hudson LG, Ding W, Wang S, Cooper KL, et al. (2004) Arsenic causes DNA damage in keratinocytes via generation of hydroxyl radicals. Chem Res Toxicol 17: 871–878.H. ShiLG HudsonW. DingS. WangKL Cooper2004Arsenic causes DNA damage in keratinocytes via generation of hydroxyl radicals.Chem Res Toxicol17871878

9. Li G, Lee LS, Li M, Tsao SW, Chiu JF (2011) Molecular changes during arsenic-induced cell transformation. J Cell Physiol. G. LiLS LeeM. LiSW TsaoJF Chiu2011Molecular changes during arsenic-induced cell transformation.J Cell Physiol

10. Huang C, Ke Q, Costa M, Shi X (2004) Molecular mechanisms of arsenic carcinogenesis. Mol Cell Biochem 255: 57–66.C. HuangQ. KeM. CostaX. Shi2004Molecular mechanisms of arsenic carcinogenesis.Mol Cell Biochem2555766

11. Ying S, Myers K, Bottomley S, Helleday T, Bryant HE (2009) BRCA2-dependent homologous recombination is required for repair of Arsenic-induced replication lesions in mammalian cells. Nucleic Acids Res 37: 5105–5113.S. YingK. MyersS. BottomleyT. HelledayHE Bryant2009BRCA2-dependent homologous recombination is required for repair of Arsenic-induced replication lesions in mammalian cells.Nucleic Acids Res3751055113

12. Lee L, Bebb G (2005) A case of Bowen's disease and small-cell lung carcinoma: long-term consequences of chronic arsenic exposure in Chinese traditional medicine. Environ Health Perspect 113: 207–210.L. LeeG. Bebb2005A case of Bowen's disease and small-cell lung carcinoma: long-term consequences of chronic arsenic exposure in Chinese traditional medicine.Environ Health Perspect113207210

13. Neuberger JS, Field RW (2003) Occupation and lung cancer in nonsmokers. Rev Environ Health 18: 251–267.JS NeubergerRW Field2003Occupation and lung cancer in nonsmokers.Rev Environ Health18251267

14. Guha Mazumder DN (2007) Arsenic and non-malignant lung disease. J Environ Sci Health A Tox. Hazard Subst Environ Eng 42: 1859–1867.DN Guha Mazumder2007Arsenic and non-malignant lung disease. J Environ Sci Health A Tox.Hazard Subst Environ Eng4218591867

15. Carmeliet P, Jain RK (2000) Angiogenesis in cancer and other diseases. Nature 407: 249–257.P. CarmelietRK Jain2000Angiogenesis in cancer and other diseases.Nature407249257

16. Folkman J (1971) Tumor angiogenesis: therapeutic implications. N Engl J Med 285: 1182–1186.J. Folkman1971Tumor angiogenesis: therapeutic implications.N Engl J Med28511821186

17. Folkman J (2006) Angiogenesis. Annu. Rev. Med 57: 1–18.J. Folkman2006Angiogenesis. Annu. Rev.Med57118

18. Voelkel NF, Douglas IS, Nicolls M (2007) Angiogenesis in chronic lung disease. Chest 131: 874–879.NF VoelkelIS DouglasM. Nicolls2007Angiogenesis in chronic lung disease.Chest131874879

19. Walters EH, Reid D, Soltani A, Ward C (2008) Angiogenesis: a potentially critical part of remodelling in chronic airway diseases? Pharmacol Ther 118: 128–137.EH WaltersD. ReidA. SoltaniC. Ward2008Angiogenesis: a potentially critical part of remodelling in chronic airway diseases?Pharmacol Ther118128137

20. Gao N, Shen L, Zhang Z, Leonard SS, He H, et al. (2004) Arsenic induces HIF-1alpha and VEGF through PI3K, Akt and reactive oxygen species in DU145 human prostate carcinoma cells. Mol Cell Biochem 255: 33–45.N. GaoL. ShenZ. ZhangSS LeonardH. He2004Arsenic induces HIF-1alpha and VEGF through PI3K, Akt and reactive oxygen species in DU145 human prostate carcinoma cells.Mol Cell Biochem2553345

21. Skinner HD, Zhong XS, Gao N, Shi X, Jiang BH (2004) Arsenic induces p70S6K1 activation and HIF-1alpha expression in prostate cancer cells. Mol Cell Biochem 255: 19–23.HD SkinnerXS ZhongN. GaoX. ShiBH Jiang2004Arsenic induces p70S6K1 activation and HIF-1alpha expression in prostate cancer cells.Mol Cell Biochem2551923

22. Meng D, Wang X, Chang Q, Hitron A, Zhang Z, et al. (2010) Arsenic promotes angiogenesis in vitro via a heme oxygenase-1-dependent mechanism. Toxicol Appl. Pharmacol 244: 291–299.D. MengX. WangQ. ChangA. HitronZ. Zhang2010Arsenic promotes angiogenesis in vitro via a heme oxygenase-1-dependent mechanism. Toxicol Appl.Pharmacol244291299

23. Mousa SA, O'Connor L, Rossman TG, Block E (2007) Pro-angiogenesis action of arsenic and its reversal by selenium-derived compounds. Carcinogenesis 28: 962–967.SA MousaL. O'ConnorTG RossmanE. Block2007Pro-angiogenesis action of arsenic and its reversal by selenium-derived compounds.Carcinogenesis28962967

24. Straub AC, Klei LR, Stolz DB, Barchowsky A (2009) Arsenic requires sphingosine-1-phosphate type 1 receptors to induce angiogenic genes and endothelial cell remodeling. Am J Pathol 174: 1949–1958.AC StraubLR KleiDB StolzA. Barchowsky2009Arsenic requires sphingosine-1-phosphate type 1 receptors to induce angiogenic genes and endothelial cell remodeling.Am J Pathol17419491958

25. Jiang BH, Zheng JZ, Aoki M, Vogt PK (2000) Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of vascular endothelial growth factor in endothelial cells. Proc Natl Acad Sci U S A 97: 1749–1753.BH JiangJZ ZhengM. AokiPK Vogt2000Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of vascular endothelial growth factor in endothelial cells.Proc Natl Acad Sci U S A9717491753

26. Xia C, Meng Q, Liu LZ, Rojanasakul Y, Wang XR, et al. (2007) Reactive oxygen species regulate angiogenesis and tumor growth through vascular endothelial growth factor. Cancer Res 67: 10823–10830.C. XiaQ. MengLZ LiuY. RojanasakulXR Wang2007Reactive oxygen species regulate angiogenesis and tumor growth through vascular endothelial growth factor.Cancer Res671082310830

27. Liu LZ, Hu XW, Xia C, He J, Zhou Q, et al. (2006) Reactive oxygen species regulate epidermal growth factor-induced vascular endothelial growth factor and hypoxia-inducible factor-1alpha expression through activation of AKT and P70S6K1 in human ovarian cancer cells. Free Radic Biol Med 41: 1521–1533.LZ LiuXW HuC. XiaJ. HeQ. Zhou2006Reactive oxygen species regulate epidermal growth factor-induced vascular endothelial growth factor and hypoxia-inducible factor-1alpha expression through activation of AKT and P70S6K1 in human ovarian cancer cells.Free Radic Biol Med4115211533

28. Semenza GL (2003) Targeting HIF-1 for cancer therapy. Nat Rev Cancer 3: 721–732.GL Semenza2003Targeting HIF-1 for cancer therapy.Nat Rev Cancer3721732

29. Forsythe JA, Jiang BH, Iyer NV, Agani F, Leung SW, et al. (1996) Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol 16: 4604–4613.JA ForsytheBH JiangNV IyerF. AganiSW Leung1996Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1.Mol Cell Biol1646044613

30. Pi J, He Y, Bortner C, Huang J, Liu J, et al. (2005) Low level, long-term inorganic arsenic exposure causes generalized resistance to apoptosis in cultured human keratinocytes: potential role in skin co-carcinogenesis. Int J Cancer 116: 20–26.J. PiY. HeC. BortnerJ. HuangJ. Liu2005Low level, long-term inorganic arsenic exposure causes generalized resistance to apoptosis in cultured human keratinocytes: potential role in skin co-carcinogenesis.Int J Cancer1162026

31. Stohrer G (1991) Arsenic: opportunity for risk assessment. Arch Toxicol 65: 525–531.G. Stohrer1991Arsenic: opportunity for risk assessment.Arch Toxicol65525531

32. Ding W, Hudson LG, Liu KJ (2005) Inorganic arsenic compounds cause oxidative damage to DNA and protein by inducing ROS and RNS generation in human keratinocytes. Mol Cell Biochem 279: 105–112.W. DingLG HudsonKJ Liu2005Inorganic arsenic compounds cause oxidative damage to DNA and protein by inducing ROS and RNS generation in human keratinocytes.Mol Cell Biochem279105112

33. Cooper KL, Liu KJ, Hudson LG (2009) Enhanced ROS production and redox signaling with combined arsenite and UVA exposure: contribution of NADPH oxidase. Free Radic Biol Med 47: 381–388.KL CooperKJ LiuLG Hudson2009Enhanced ROS production and redox signaling with combined arsenite and UVA exposure: contribution of NADPH oxidase.Free Radic Biol Med47381388

34. Sampayo-Reyes A, Hernandez A, El Yamani N, Lopez-Campos C, Mayet-Machado E, et al. (2010) Arsenic induces DNA damage in environmentally exposed Mexican children and adults. Influence of GSTO1 and AS3MT polymorphisms. Toxicol Sci 117: 63–71.A. Sampayo-ReyesA. HernandezN. El YamaniC. Lopez-CamposE. Mayet-Machado2010Arsenic induces DNA damage in environmentally exposed Mexican children and adults. Influence of GSTO1 and AS3MT polymorphisms.Toxicol Sci1176371

35. Connolly DT, Heuvelman DM, Nelson R, Olander JV, Eppley BL, et al. (1989) Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J Clin Invest 84: 1470–1478.DT ConnollyDM HeuvelmanR. NelsonJV OlanderBL Eppley1989Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis.J Clin Invest8414701478

36. Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N (1989) Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 246: 1306–1309.DW LeungG. CachianesWJ KuangDV GoeddelN. Ferrara1989Vascular endothelial growth factor is a secreted angiogenic mitogen.Science24613061309

37. Kim KJ, Li B, Winer J, Armanini M, Gillett N, et al. (1993) Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo. Nature 362: 841–844.KJ KimB. LiJ. WinerM. ArmaniniN. Gillett1993Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo.Nature362841844

38. Kierszniewska-Stepien D, Pietras T, Gorski P, Stepien H (2006) Serum vascular endothelial growth factor and its receptor level in patients with chronic obstructive pulmonary disease. Eur Cytokine Netw 17: 75–79.D. Kierszniewska-StepienT. PietrasP. GorskiH. Stepien2006Serum vascular endothelial growth factor and its receptor level in patients with chronic obstructive pulmonary disease.Eur Cytokine Netw177579

39. Yang Q, Underwood MJ, Hsin MK, Liu XC, He GW (2008) Dysfunction of pulmonary vascular endothelium in chronic obstructive pulmonary disease: basic considerations for future drug development. Curr Drug Metab 9: 661–667.Q. YangMJ UnderwoodMK HsinXC LiuGW He2008Dysfunction of pulmonary vascular endothelium in chronic obstructive pulmonary disease: basic considerations for future drug development.Curr Drug Metab9661667

40. Jatta K, Eliason G, Portela-Gomes GM, Grimelius L, Caro O, et al. (2009) Overexpression of von Hippel-Lindau protein in skeletal muscles of patients with chronic obstructive pulmonary disease. J Clin Pathol 62: 70–76.K. JattaG. EliasonGM Portela-GomesL. GrimeliusO. Caro2009Overexpression of von Hippel-Lindau protein in skeletal muscles of patients with chronic obstructive pulmonary disease.J Clin Pathol627076

41. Hasaneen NA, Zucker S, Lin RZ, Vaday GG, Panettieri RA, et al. (2007) Angiogenesis is induced by airway smooth muscle strain. Am J Physiol Lung Cell Mol Physiol 293: L1059–L1068.NA HasaneenS. ZuckerRZ LinGG VadayRA Panettieri2007Angiogenesis is induced by airway smooth muscle strain.Am J Physiol Lung Cell Mol Physiol293L1059L1068

42. Kamat CD, Green DE, Curilla S, Warnke L, Hamilton JW, et al. (2005) Role of HIF signaling on tumorigenesis in response to chronic low-dose arsenic administration. Toxicol. Sci. 86: 248–257.CD KamatDE GreenS. CurillaL. WarnkeJW Hamilton2005Role of HIF signaling on tumorigenesis in response to chronic low-dose arsenic administration.Toxicol. Sci.86248257

43. Mottet D, Michel G, Renard P, Ninane N, Raes M, et al. (2003) Role of ERK and calcium in the hypoxia-induced activation of HIF-1. J Cell Physiol 194: 30–44.D. MottetG. MichelP. RenardN. NinaneM. Raes2003Role of ERK and calcium in the hypoxia-induced activation of HIF-1.J Cell Physiol1943044

44. Zhong H, Chiles K, Feldser D, Laughner E, Hanrahan C, et al. (2000) Modulation of hypoxia-inducible factor 1alpha expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics. Cancer Res 60: 1541–1545.H. ZhongK. ChilesD. FeldserE. LaughnerC. Hanrahan2000Modulation of hypoxia-inducible factor 1alpha expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics.Cancer Res6015411545

45. Zhou Q, Liu LZ, Fu B, Hu X, Shi X, et al. (2007) Reactive oxygen species regulate insulin-induced VEGF and HIF-1alpha expression through the activation of p70S6K1 in human prostate cancer cells. Carcinogenesis 28: 28–37.Q. ZhouLZ LiuB. FuX. HuX. Shi2007Reactive oxygen species regulate insulin-induced VEGF and HIF-1alpha expression through the activation of p70S6K1 in human prostate cancer cells.Carcinogenesis282837