Arsenic-Induced Cell Transformation and Angiogenesis Requires NADPH Oxidase Isoform 2-Derived Reactive Oxygen Species
Arsenic is a naturally-occurring element present throughout the earth's crust. Humans are exposed to arsenic by occupational means, such as mining and smelting of metal ores, and by non-occupational means, such as consumption of arsenic-contaminated water and food grown in contaminated soil. It is estimated that tens of millions of people throughout the world are exposed to arsenic at levels greater than recommended by the U.S. Environmental Protection Agency (EPA) and World Health Organization (WHO). Exposure to arsenic for long periods leads to increased risk for cancer of the lung, skin, and bladder among others. Arsenic is considered a carcinogen by several regulatory agencies including the U.S. EPA, the National Toxicology Program, and the International Agency for Research on Cancer. Despite a clear connection between arsenic exposure and cancer, cellular mechanisms for arsenic in inducing cell transformation and tumorigenesis has not been established.
In order to study the mechanism of arsenic-induced cell transformation, lung epithelial cells were exposed to different concentrations of arsenic for 26 weeks. These arsenic-transformed (As-T) cells displayed qualities of transformed cells such as increased proliferation rates, increased growth independent of attachment, and formation of tumors in vivo. As-T cells were also observed to have increased activation of AKT and ERK as well as higher levels of reactive oxygen species (ROS). Interruption of AKT and ERK signaling resulted in decreased cell proliferation and anchorage-independent growth. Suppression of ROS levels by catalase reduced the activation of both AKT and ERK, reduced proliferation and anchorage-independent growth, and reduced tumor growth.
These results suggest that arsenic promotes oxidative stress to induce cell transformation. However, the cellular mechanism of ROS generation is not well understood. We found that chemical and peptide inhibitors specific to NADPH oxidase reduced ROS levels in As-T cells. Immunoblotting revealed that As-T cells have increased protein expression of NADPH oxidase isoform 2 (NOX2) and its corresponding activating subunits p47phox and p67phox. Silencing RNA-mediated knockdown of NOX2 or p47 phox lowered ROS levels in As-T cells. Immunoprecipitation experiments demonstrated increased phosphorylation of p47phox in As-T cells, a critical step in the activation of NOX2. Evidence also indicates increased activation of Rac1 in As-T cells, another critical subunit to the active NOX2 complex. As-T cells also exhibited increased NADPH oxidase activity compared with control cells. Knockdown of NOX2 or p47phox resulted in reduced activation of AKT and ERK. Knockdown of NOX2 or p47phox also reduced As-T proliferation and anchorage-independent growth. These results suggest that NOX2-derived ROS regulate arsenic-induced cell transformation.
Previously published literature and preliminary data to this project suggest arsenic may promote a pro-angiogenic environment. To further study this, both acute As exposure and As-T cells were shown to induce in vivo angiogenesis greater than control cells. Evidence also indicated increased VEGF and IL-8 production by As-T cells. Knockdown of VEGF or IL-8 in As-T cells compromised the ability of these cells to promote angiogenesis. Levels of VEGF and IL-8 mRNA in As-T cells were significantly reduced after knockdown of NOX2 or p47 phox. Furthermore, angiogenesis induced by acute As exposure or by As-T cells was reduced with knockdown of NOX2. These data suggest that ROS derived from NOX2 are critical for cells for inducing angiogenesis.
Together these studies suggest that ROS are critical mediators of cell transformation by arsenic. Further investigation revealed arsenic upregulates NOX2, which enhances ROS generation and promotes cell transformation and tumor angiogenesis.