Overexpression of CYP4A1‐20‐HETE in U251 Glioma Cell Induces Hyperproliferative Phenotypes in vitro and in vivo

Abstract

Cytochrome P450 (CYP) omega hydroxylases such as CYP 4A1 and 4F enzymes convert arachidonic acid to 20‐HETE. 20‐HETE is a potent stimulator of the growth of human glioma cells in vitro. Herein, we report that increasing the endogenous generation of 20‐HETE by overexpressing CYP4A1 in U251 results in a ∼ 2 fold higher proliferation rate than control U251. This is accompanied by an increased S phase in the cell. U251 O show a disorganized and disrupted cytoskeleton, and have constitutive activation of phosphorylated ERK1/2 and increased expression of cyclin D1/2. The hyperproliferative and signaling effects in U251 O are abolished by the selective CYP4A inhibitor HET0016 and by the putative 20‐HETE antagonist 20‐HEDE. This demonstrates that the hyperproliferative state is caused by the high endogenous production of 20‐HETE by CYP4A1. Dihydroethidium assay show that the U251 O cells have marked increases in superoxide formation. Treatment with PEG‐SOD abolished the hyperproliferative state, indicating the crucial contribution of superoxide to the hyperproliferative phenotype. In vivo, MRI studies showed that the tumor of the rats implanted with U251 O had a ∼10 fold higher tumor volume compared with U251. High 20‐HETE leads to enhanced proliferation and to a fast‐growing tumor in vivo. These findings suggest that 20‐HETE synthases and 20‐HETE could play an important role in growth regulation of human gliomas.