9L Gliosarcoma Cell Proliferation and Tumor Growth in Rats Are Suppressed by N-Hydroxy-N′-(4-butyl-2-methylphenol) Formamidine (HET0016), a Selective Inhibitor of CYP4A

Abstract

The present study examined the effects of N-hydroxy-NЈ-(4-butyl-2 methylphenyl) formamidine (HET0016), a selective inhibitor of the formation of 20-hydroxyeicosatrienoic acid (20-HETE) on the growth of 9L rat gliosarcoma cells in vitro and in vivo. After 48 h of incubation, HET0016 reduced the proliferation of 9L in vitro by 55%, and this was associated with a fall in p42/p44 mitogen-activated protein kinase and stress-activated protein kinase/c-Jun NH 2 -terminal kinase phosphorylation and increased apoptosis. HET0016 inhibited epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)-induced proliferation and diminished phosphorylation of PDGF receptors. A stable 20-HETE analog increased 9L cell proliferation. In vivo, chronic administration of HET0016 (10 mg/kg/day i.p.) for 2 weeks reduced the volume of 9L tumors by 80%. This was accompanied by a 4-fold reduction in the mitotic index, a 3-to 4-fold increase in the apoptotic index, and a ϳ50% decrease in vascularization in the tumor. HET0016 treatment increased mean survival time of the animals from 17 to 22 days. Liquid chromatography/mass spectrometry experiments indicated that neither 9L cells grown in vitro nor 9L tumors removed produce 20-HETE when incubated with arachidonic acid. The normal surrounding brain tissue, however, avidly makes 20-HETE, and this activity is selectively inhibited by HET0016. These results suggest that HET0016 may be the prototype of a class of antigrowth compounds that may be efficacious for treating malignant brain tumors. In vivo, it may act in part by inhibiting the formation of 20-HETE by the surrounding tissue. However, the antiproliferative effects of HET0016 on 9L cells in vitro seem unrelated to its ability to inhibit the formation of 20-HETE.

20-HETE increases survival and decreases apoptosis in pulmonary arteries and pulmonary artery endothelial cells

“…Guo et al (14) described proliferative, but not antiapoptotic effects of 20-HETE mediated by VEGF in human dermal microvascular cells. In a separate study, however, this group reported antiproliferative effects of HET0016 (an inhibitor of the formation of 20-HETE) in gliosarcoma cells in vivo and in vitro (15). Our studies support a pro-proliferative effect of 20-HETE in BPAECs, but we focused on the mechanisms through which this lipid promotes survival in these cells.…”

The Cytochrome P450 4A/F-20-Hydroxyeicosatetraenoic Acid System: A Regulator of Endothelial Precursor Cells Derived from Human Umbilical Cord Blood

“…Proliferation studies were performed as described previously (Guo et al, , 2006. EPCs were incubated with VEGF (20 ng/ml) or 20-HETE (1 M) for 48 h. In some experiments, the cultures were exposed to VEGF in the presence of 10 M N-hydroxy-NЈ-(4-butyl-2-methylphenyl)formamidine (HET0016), a selective inhibitor of 20-HETE synthesis.…”

20-HETE activates the Raf/MEK/ERK pathway in renal epithelial cells through an EGFR- and c-Src-dependent mechanism

“…We and others found that inhibition of 20-HETE synthesis reduced ERK1/2 activation (9,10,28,29), but the mechanism by which 20-HETE stimulated this protein complex remains to be established. Studies have implicated an interaction between receptor and nonreceptor tyrosine kinases to mediate the functions of 20-HETE in the kidney (7,39).…”

“…These tyrosine kinases, specifically EGF receptor (EGFR), are known to play an important role in mediating growth and cell proliferation through the activation of the Raf/MEK/ERK pathway in PKD and various epithelial cancers (22,33,40,46). To further validate the potential interaction of 20-HETE with tyrosine kinases, recent studies showed that inhibition of 20-HETE synthesis reduced EGFR activation, which was associated with reduced proliferative activity (9,10,28).…”