Inhibition of CYP4A decreases 9L gliosarcoma cell proliferation and brain tumor volume in rats

1066 Cytochrome P450 4A (CYP4A) is a member of the CYP family of fatty acid metabolizing enzyme. CYP4A metabolizes arachidonic acid to 20-HETE, a potent vasoconstrictor. We have previously reported that a highly selective inhibitor of CYP4A markedly reduces the basal growth rate of a human glioblastoma cell line, U251. We hypothesized that a CYP4A metabolite, presumably 20-HETE, plays an important role in regulating the growth of an aggressive cancer cell line, 9L rat gliosarcoma. In this study, we examined the effects of a selective inhibitor of CYP4A, HET0016 on the growth of 9L in vitro and in 9L-induced brain tumors in rats in vivo . CYP4A genes are expressed in 9L cells as determined by RT-PCR. Inhibition of CYP4A activity with the highly selective inhibitor, HET0016, (N-hydroxy-N’-(4-butyl-2 methylphenyl) formamidine) decreased the basal growth rate of 9L cells in a dose-related manner. Addition of 10 μM HET0016 reduced the proliferation of 9L cells after 48 hr by 60%. Addition of 100 μM DDMS (N-methylsulfonyl-12, 12-dibromododec-11-enamide), a structurally different inhibitor of CYP4A enzymes, also inhibited 9L cell proliferation by 60%. A stable 20-HETE agonist (20-hydroxyeicosa-5(Z), 14(Z)-dienoic acid, WIT003) at 1 μM rescued HET0016 inhibited cell proliferation by ∼60%. Epidermal growth factor (EGF; 200 ng/ml) increased the proliferation of 9L cells grown in vitro by ∼30%. A similar degree of stimulation was obtained with 1 μM WIT003. HET0016 almost abolished EGF-induced growth of 9L cells. Western blot analysis showed that HET0016 decreases the phosphorylation of p42/p44 MAPK and SAPK/JNK, suggesting that the inhibitors are interfering with the regulation of growth signaling. In a second series of experiments, brain tumors were induced by injecting 9L cells directly in the forebrain of rats followed by intraperitoneal daily injections of HET0016 (10 mg/Kg/day) for ∼2 weeks. Injection significantly reduced the volume of the brain tumor by ∼80%, which is resulted from a decreased mitosis and an increased apoptosis of tumor cells. An increased survival of ∼5 days was recorded in HET0016-injected rats compared to the control animals. These results suggest that CYP4A and presumably 20-HETE are involved in the regulation of proliferation of rat 9L gliosarcoma cells both in vitro and in vivo , and that inhibitors of CYP4A enzymes may be useful as adjuvant in treatment of malignant gliosarcoma tumors.