Effects And Mechanisms Of Epoxyeicosatrienoic Acids On Reactive Oxygen Species Generation In Endothelial Cells And Tumor Cells

BackgroundCytpchrome P450 (CYP) epoxygenases convert arachidonic acid to four epoxyeicosatrienoic acid (EET) regioisomers, 5,6-, 8,9-, 11,12-, and 14,15-EET, that function as the critical lipid mediators involved in several biological events in human body. EETs play a crucial role in cardiovascular homeostasis that have been considered including mechanisms 1) to preserve endothelial function by increasing NO bioavailability, 2) to inhibit the expression of inflammation and adhesion factors in endothelial cells, 3) to promote endothelial cells proliferation and angiogenesis, 4) to inhibit vascular smooth muscle cells (VSMC) proliferation and migration, 5) to increase fibrolysis. These evidences indicated that EET extensively protect the cardiovascular system. Oxidative stress has been associated with diverse pathophysiological events, including neurodegeneration, diabetes mellitus, renal disease and cancer. Reactive oxygen species (ROS) elicit direct cellular damage and mitogenicity, serve as intracellular second messengers, scavenge vasoprotective NO, increase inflammatory cells adhesion, and promote VSMC proliferation and migration. Recently, it has been verified ROS also play a critical role in the incidence and development of vasculopathies, including atherosclerosis, hypertension and restenosis after angioplasty. Now that the EET maintained cardiovascular homeostasis, whereas ROS has been served as the "unstable factors", whether or not EET reduces ROS injury in vascular walls is not clear. Investigating the action of EET on oxidative stress and ROS generation in cardiovascular system could definite the physiological role of EET and provide the theoretical basis and the target for the prevention and treatment of cardiovascular diseases. At the meantime, we revealed for the first time that EETs promote human tumor cells proliferation and metastasis, and inhibit tumor cells apoptosis. ROS are the critical regulating molecules of apoptosis in tumor cells, and extensive evidence indicates that ROS plays a piotal role in tumor cell apoptosis whether induced by ischemia, drugs, or receptor mediated factors. For this reason, we investigated whether EET inhibits tumor cells apoptosis correlated with ROS generation.MethodsAfter treating with EET for 24 hours, bovine arotic endothelial cells (BAECs) was treated with angiotensin II (Ang II) for 1 hour. The generation of ROS was measured by flowcytometry. The expression of NAD(P)H oxidase subunits, superoxide dismutases (SOD) and catalase (CAT) were detected by western blot. The activity of SOD and CAT were measured by biochemical assays. Following cytoplasmic and membrane protein separation, the NAD(P)H oxidase subunit p47phox and Rac1 GTPase translocation were detected by western blot. ROS and apoptosis were induced by arsenic trioxide (ATO) in Tca-8113 human tumor cells, and were measured by flowcytometry. Caspase activity was measured by colorimetry assay. Mitochondrial membrane potential was measured by JC-1 assay. The involved cell signal molecular changes were detected by western blot.Results1. EETs alleviate reactive oxygen species generation in BAECs and the mechanisms24 hours pretreatment of EETs significantly inhibited Ang II-induced ROS generation in BAECs. This effect may be mediated by inhibiting NAD (P) H oxidase subunits: gp91phox expression, reduced translocation of p47phox and rac1 and by increasing SOD expression and its activity. 2. EETs reduce reactive oxygen production in tumor cells and the mechanismsEETs can inhibit the tumor cells reactive oxygen generation and apoptosis induced by ATO. EETs can also attenuate the loss of mitochondrial membrane potential, caspase 9 and 3 activation as well as P38, JNK signaling pathway activation caused by ATO. Inhibition of CYP2J2 activity cooperated with ATO increased ROS generation and apoptosis in tumor cells.ConclusionUsing molecular and cellular biology techniques, we investigated the regulation of reactive oxygen by EETs and the mechanisms in vitro in cultured endothelial cells and tumor cells. We found that:1. EETs inhibited Ang II-induced reactive oxygen species generation through inhibiting NAD(P)H oxidase subunits expression and translocation and through increasing SOD expression as well as its activity. These effects may dependent on AMPK, PPAR-γ, ERK signaling pathway.2. EETs also inhibited the ROS generation induced by ATO in tumor cells. Moreover, EETs can inhibit ROS-mediated mitochondrial pathway of apoptosis, which enriched the mechanisms of EETs act on tumor cell apoptosis. Meanwhile, inhibition of CYP2J2 enhanced the effect of ATO on tumor cells, which raise a new strategy for the prevention and treatment of clinical oncology.