Reactive oxygen species and angiogenesis: NADPH oxidase as target for cancer therapy

Abstract

Angiogenesis is essential for tumor growth, metastasis, arteriosclerosis as well as embryonic development and wound healing. Its process is dependent on cell proliferation, migration and capillary tube formation in endothelia cells (ECs). High levels of reactive oxygen species (ROS) such as superoxide and H2O2 are observed in various cancer cells. Accumulating evidence suggests that ROS function as signaling molecules to mediate various growth-related responses including angiogenesis. ROS-dependent angiogenesis can be regulated by endogenous antioxidant enzymes such as SOD and thioredoxin. Vascular endothelial growth factor (VEGF), one of the major angiogenesis factor, is induced in growing tumors and stimulates EC proliferation and migration primarily through the VEGF receptor type2 (VEGFR2, Flk1/KDR). Major source of ROS in ECs is a NADPH oxidase which consists of Nox1, Nox2, Nox4, Nox5, p22phox, p47phox and the small G-protein Rac1. NADPH oxidase is activated by various growth factors including VEGF and angiopoietin-1 as well as hypoxia and ischemia, and ROS derived from this oxidase are involved in VEGFR2 autophosphorylation, and diverse redox signaling pathways leading to induction of transcription factors and genes involved in angiogenesis. Dietary antioxidants appear to be effective for treatment of tumor angiogenesis. The aim of this review is to provide an overview of the recent progress on role of ROS derived from NADPH oxidase and redox signaling events involved in angiogenesis. Understanding these mechanisms may provide insight into the NADPH oxidase and redox signaling components as potential therapeutic targets for tumor angiogenesis.

Figure 1. Anti-angiogenic therapy targeting NADPH oxidase (ROS)-dependent VEGF signaling

Progression to a growing tumor is characterized by induction of proangiogenic factors such as vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPs), and VEGF receptors (VEGFR) in the growing endothelial cells (ECs). VEGF is induced from tumor and stimulates permeability, proliferation, migration and tube formation of ECs primarily through the VEGFR type2 (VEGFR2). The conversion to the angiogenic phenotype in previously dormant tumors is known as the “angiogenic switch”. Thus, anti-angiogenic therapy is essential strategy for the treatment of tumor. In addition to VEGF and VEGFR2, ROS and NADPH oxidase are potential therapeutic targets for the treatment of tumor angiogenesis.

Figure 2. A, Shematic diagram of the structure of NADPH oxidase in ECs

gp91^phox (Nox2) and its homologues (Nox1, Nox4 and Nox5) and cytosolic components p47^phox, p67^phox and small GTPase Rac1 have been identified in ECs. B, Transmembrane topology of Nox and Duox enzymes. The predicted transmembrane α-helices contain conserved histidine residues which comprise binding sites for haems. The carboxyl-terminal domain folds within the cytoplasm and binds to flavin adenine dinucleotide (FAD) and NADPH. The enzymes catalyze the transfer of electrons from NADPH to molecular oxygen, to form O₂⁻ across the membrane. The amino-terminal calcium-binding domain of Nox5 and Duox enzymes are also predicted to be on the cytosolic side of the membrane. Additional transmembrane α-helix of the Duox enzymes at the amino-terminus localize the peroxidase domain to the opposite side of the membrane, where it can use ROS generated by the catalytic core to generate more powerful oxidant species that then oxidize extracellular substrates.

Figure 3. Role of ROS derived from NADPH oxidase in VEGF signaling linked to induction of transcription factors and genes involved in angiogenesis

Ischemia/hypoxia stimulates induction of VEGF which stimulates NADPH oxidase to produce ROS, thereby inducing oxidative inactivation of protein tyrosine phosphatases (PTPs) and PTEN to promote VEGFR2 autophosphorylation and downstream redox signaling events or directly activating redox signaling kinases. These events are converged and integrated to induce various redox sensitive transcriptional factors and gene expression, which are involved in angiogenesis.