Expression of CYP4A1 in U251 human glioma cell induces hyperproliferative phenotype in vitro and rapidly growing tumors in vivo.
Exogenous 20-hydroxyeicosatetraenoic acid (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 2-fold (p < 0.01). Compared with 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 extracellular signal-regulated kinase 1/2, cyclin D1/2, and vascular endothelial growth factor 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], by small interfering RNA against the enzyme, and by the putative 20-HETE antagonist, 20-hydroxyeicosa-5(Z),14(Z)-dienoic acid. In vivo, implantation of U251O cells in the brain of nude rats resulted in a approximately 10-fold larger tumor volume (10 days postimplantation) 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. Further studies are needed to determine whether 20-HETE plays a role promoting growth of some human gliomas.
Guo
Austin M
AM
Henry Ford Hospital, Detroit, Michigan 48202, USA. mguo1@hfhs.org
Sheng
Ju
J
Scicli
Gloria M
GM
Arbab
Ali S
AS
Lehman
Norman L
NL
Edwards
Paul A
PA
Falck
John R
JR
Roman
Richard J
RJ
Scicli
A Guillermo
AG
D016718:Arachidonic Acid / Q000378:metabolism*; D002448:Cell Adhesion; D002453:Cell Cycle; D049109:Cell Proliferation; D042926:Cytochrome P-450 CYP4A / Q000502:physiology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000502:physiology; D005910:Glioma / Q000378:metabolism* / Q000473:pathology*; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000096:biosynthesis*; D008279:Magnetic Resonance Imaging; D014411:Neoplastic Stem Cells; D018384:Oxidative Stress; D010641:Phenotype; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000502:physiology
D006893:Hydroxyeicosatetraenoic Acids; D042461:Vascular Endothelial Growth Factor A; D016718:Arachidonic Acid; C055987:20-hydroxy-5,8,11,14-eicosatetraenoic acid; D042926:Cytochrome P-450 CYP4A; D048049:Extracellular Signal-Regulated MAP Kinases