Expression of CYP4A1 in U251 human glioma cell induces hyperproliferative phenotype in vitro and rapidly growing tumors in vivo

Austin M. Guo, Ju Sheng, Gloria M. Scicli, Ali S. Arbab, Norman L. Lehman, Paul A. Edwards, John R. Falck, Richard J. Roman, A. Guillermo Scicli

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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.

Original languageEnglish (US)
Pages (from-to)10-19
Number of pages10
JournalJournal of Pharmacology and Experimental Therapeutics
Volume327
Issue number1
DOIs
StatePublished - Oct 1 2008

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Glioma
Phenotype
Neoplasms
Growth
Cytochrome P-450 CYP4A
Nude Rats
Staining and Labeling
Vinculin
20-hydroxy-5,8,11,14-eicosatetraenoic acid
In Vitro Techniques
Mitogen-Activated Protein Kinase 3
Mitogen-Activated Protein Kinase 1
Cyclin D1
Tumor Burden
Cytoskeleton
Vascular Endothelial Growth Factor A
Small Interfering RNA
Oxidative Stress
Complementary DNA
Cell Culture Techniques

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

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Expression of CYP4A1 in U251 human glioma cell induces hyperproliferative phenotype in vitro and rapidly growing tumors in vivo. / Guo, Austin M.; Sheng, Ju; Scicli, Gloria M.; Arbab, Ali S.; Lehman, Norman L.; Edwards, Paul A.; Falck, John R.; Roman, Richard J.; Scicli, A. Guillermo.

In: Journal of Pharmacology and Experimental Therapeutics, Vol. 327, No. 1, 01.10.2008, p. 10-19.

Research output: Contribution to journalArticle

Guo, Austin M. ; Sheng, Ju ; Scicli, Gloria M. ; Arbab, Ali S. ; Lehman, Norman L. ; Edwards, Paul A. ; Falck, John R. ; Roman, Richard J. ; Scicli, A. Guillermo. / Expression of CYP4A1 in U251 human glioma cell induces hyperproliferative phenotype in vitro and rapidly growing tumors in vivo. In: Journal of Pharmacology and Experimental Therapeutics. 2008 ; Vol. 327, No. 1. pp. 10-19.
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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.",
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AU - Lehman, Norman L.

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