Received for publication May 14, 2008.
Revised June 27, 2008.
Accepted for publication June 27, 2008.

Expression of CYP4A1 in U251 Human Glioma Cell Induces Hyperproliferative Phenotype in vitro and Rapidly Growing Tumors in vivo

Abstract

Exogenous 20-HETE increases the growth of human glioma cells in vitro. However, glioma cells in culture show negligible 20-HETE synthesis. We examined whether inducing the expression of a 20-HETE synthase in a human glioma U251 cell line would increase proliferation. U251 cells transfected with CYP4A1 cDNA (termed U251 O) increased the formation of 20-HETE from less than 1 to over 60 pmol/min/mg proteins and increased their proliferation rate by two fold (p < 0.01). Compared to control U251, U251 O cells were rounded, smaller, showed a disorganized cytoskeleton, exhibited reduced vinculin staining, and were easily detached from the growing surface. They showed a marked increase in dihydroethidium staining suggesting increased oxidative stress. The expression of phosphorylated ERK1/2, cyclin D1/2 and VEGF was markedly elevated in U251 O. The hyperproliferative and signaling effects seen in U251 O cells are abolished by selective CYP4A inhibition of 20-HETE formation with HET0016 (N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine) as well as by siRNA against the enzyme, and by the putative 20-HETE antagonist, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE). In vivo, implantation of U251O cells in the brain of nude rats resulted in a ~10 fold larger tumor volume (10 days post implantation) as compared with animals receiving mock-transfected U251 cells. These data show that elevations in 20-HETE synthesis in U251 cells lead to an increased growth both in vitro and in vivo. This suggests that 20-HETE may have proto-oncogenic properties in U251 human gliomas and contributes to the regulation of the growth of some human gliomas.

Key words: 20-HETE synthase, Glioma, arachidonic acid metabolite, cytochrome P450 4A, growth, superoxide