Alpha-difluoromethylornithine (DFMO) as a potent arginase activity inhibitor in human colon carcinoma cells.
Alpha-difluoromethylornithine (DFMO) is commonly used as a specific ornithine decarboxylase (ODC, EC4.1.1.17) irreversible inhibitor. ODC is the enzyme responsible for polyamine biosynthesis, which has been shown to be strictly necessary for cell proliferation. In HT-29 Glc-/+ cells, L-arginine is the major precursor of these molecules through the sequential actions of arginase, which leads to L-ornithine generation and ODC. L-ornithine, a substrate for ODC, retroinhibits arginase. Since DFMO is an ornithine analogue, we searched for a direct effect of this agent upon arginase. The flux of L-arginine through arginase in intact cells was inhibited by 51+/-11% by 10 mM of DFMO whereas 10 mM of L-valine, a known potent arginase inhibitor, inhibited this flux by 73+/-6%. DFMO equilibrated between extracellular and intercellular spaces and, when used at 10-mM concentration, was without effect on L-arginine net uptake. Measurement of arginase activity in HT-29 cell homogenates with increasing concentrations of DFMO and L-arginine led to an inhibition with a calculated Ki (inhibitory constant) equal to 3.9+/-1.0 mM. L-ornithine was less effective than DFMO in inhibiting arginase activity. Bovine liver arginase, used as another source of the enzyme, was also severely inhibited by DFMO. The inhibitory effect of DFMO upon arginase, one step upstream of the ODC reaction in the metabolic conversion of L-arginine to polyamines, is of potential physiological importance, since it could alter the production of ornithine and thus its metabolism in pathways other than the ODC pathway.
Laboratoire de Nutrition et de Sécurité Alimentaire, Institut National de la Recherche Agronomique, Jouy-en-Josas, France.
M
M
Selamnia
C
C
Mayeur
V
V
Robert
F
F
Blachier
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001119:Arginase / Q000037:antagonists & inhibitors*; D001120:Arginine / Q000378:metabolism; D002417:Cattle; D000518:Eflornithine / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D019073:HT29 Cells; D006801:Humans; D008099:Liver / Q000201:enzymology; D009952:Ornithine / Q000378:metabolism; D065108:Ornithine Decarboxylase Inhibitors
D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D065108:Ornithine Decarboxylase Inhibitors; D001120:Arginine; D009952:Ornithine; D001119:Arginase; D000518:Eflornithine