Abstract
The present study examined the effects of N-hydroxy-N′-(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 NH2-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 ∼50% 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.
Footnotes
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This work was supported by National Institutes of Health Grants EY014385 (A.G.S.), GM31278 (J.R.F.), and HL36279, HL059996, and HL29587 (R.J.R.), and from the Robert A. Welch Foundation to J.R.F.
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This work was presented in part at 2005 American Association for Cancer Research annual meeting in Anaheim, CA, on April 16–20, 2005.
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doi:10.1124/jpet.105.097782.
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ABBREVIATIONS: EGF, epidermal growth factor; FGF, fibroblast growth factor; VEGF, vascular endothelial growth factor; 20-HETE, 20-hydroxyeicosatetraenoic acid; MAPK, mitogen-activated protein kinase; HET0016, N-hydroxy-N′-(4-butyl-2-methylphenol) formamidine; EGFR, EGF receptor; DMEM, Dulbecco's minimal essential medium; PDGF, platelet-derived growth factor; WIT003, 20-hydroxyeicosa-5(Z), 14(Z)-dienoic acid; EtOH, ethanol; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; PI, propidium iodide; SAPK/JNK, stress-activated protein kinase/c-Jun NH2-terminal kinase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; VWF, von Willebrand factor; DAPI, 4,6-diamidino-2-phenylindole; AA, arachidonic acid; EEZE, 14,15-epoxyeicosa-5(Z)-enoic-methyl sulfonylimide; HPLC, high-performance liquid chromatography; EET, epoxyeicosatrienoic acid; LC/MS, liquid chromatography/mass spectrometry.
- Received October 28, 2005.
- Accepted December 8, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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