Abstract
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 ∼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.
Footnotes
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This study was supported by the National Institutes of Health (Grants EY014385 to A.G.S., GM31278 to J.R.F., and HL 036279 to R.J.R.) and by a grant from the Robert A. Welch Foundation (to J.R.F.).
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Part of this work were previously presented as follows: Guo AM, Edwards PA, Falck JR, Roman RJ, Jafari-Khouzani K, and Scicli GA (2008) Overexpression of CYP4A1-20-HETE in U251 glioma cell induces a hyperproliferative phenotype in vitro and rapidly growing tumors in vivo. 10th Annual Winter Eicosanoid Conference; 2008 Mar 9–12; Baltimore, MD; and Guo AM, Roman RJ, Falck JR, Jafari-Khouzani K, Edwards PA, and Scicli GM (2008) Overexpression of CYP4A1-20-HETE in U251 glioma cell induces hyperproliferative phenotypes in vitro and in vivo; 2008 Experimental Biology Annual Meeting; 2008 Apr 5–9; San Diego, CA.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.108.140889.
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ABBREVIATIONS: AA, arachidonic acid; P450, cytochrome P450; 20-HETE, 20-hydroxyeicosatetraenoic acid; VEGF, vascular endothelial growth factor; DMEM, Dulbecco's modified Eagle's medium; siRNA, small interfering RNA; HET0016, N-hydroxy-N′-(4-butyl-2-methylphenyl)-formamidine; DDMS, N-methylsulfonyl-12,12-dibromododec-11-enamide; 20-HEDE, 20-hydroxyeicosa-5(Z),14(Z)-dienoic acid; ERK, extracellular signal-regulated kinase; DHE, dihydroethidium; FITC, fluorescein isothiocyanate; DAPI, 4,6-diamidino-2-phenylindole; MRI, magnetic resonance imaging; ROI, region(s) of interest; SI, signal intensity; PEG-SOD, polyethylene glycol-superoxide dismutase; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene; ROS, reactive oxygen species; MAPK, mitogen-activated protein kinase; 6,15-HEDGE, N-[20-hydroxyeicosa-6(Z),15(Z)-dienoyl]glycine.
- Received May 13, 2008.
- Accepted June 27, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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