naphthoquinones and acivicin

The pyrimidine de novo nucleotide synthesis consists of 6 sequential steps. Various inhibitors against these enzymes have been developed and evaluated in the clinic for their potential anticancer activity: acivicin inhibits carbamoyl-phosphate-synthase-II, N-(phosphonacetyl)-L- aspartate (PALA) inhibits aspartate-transcarbamylase, Brequinar sodium and dichloroallyl-lawsone (DCL) inhibit dihydroorotate-dehydrogenase, and pyrazofurin (PF) inhibits orotate-phosphoribosyltransferase. We compared their growth inhibition against 3 cell lines from head-and-neck-cancer (HEP-2, UMSCC-14B and UMSCC-14C) and related the sensitivity to their effects on nucleotide pools. In all cell lines Brequinar and PF were the most active compounds with IC50 (50% growth inhibition) values between 0.06-0.37 µM, Acivicin was as potent (IC50s 0.26-1 µM), but DCL was 20-31-fold less active. PALA was most inactive (24-128 µM). At equitoxic concentrations, all pure antipyrimidine de novo inhibitors depleted UTP and CTP after 24 hr exposure, which was most pronounced for Brequinar (between 6-10% of UTP left, and 12-36% CTP), followed by DCL and PF, which were almost similar (6-16% UTP and 12-27% CTP), while PALA was the least active compound (10-70% UTP and 13-68% CTP). Acivicin is a multi-target inhibitor of more glutamine requiring enzymes (including GMP synthetase) and no decrease of UTP was found, but a pronounced decrease in GTP (31-72% left). In conclusion, these 5 inhibitors of the pyrimidine de novo nucleotide synthesis varied considerably in their efficacy and effect on pyrimidine nucleotide pools. Inhibitors of DHO-DH were most effective suggesting a primary role of this enzyme in controlling pyrimidine nucleotide pools.

Topics: Amides; Antineoplastic Agents; Aspartate Carbamoyltransferase; Aspartic Acid; Biphenyl Compounds; Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); Carcinoma, Squamous Cell; Cell Line, Tumor; Dihydroorotate Dehydrogenase; Head and Neck Neoplasms; Humans; Isoxazoles; Naphthoquinones; Orotate Phosphoribosyltransferase; Oxidoreductases Acting on CH-CH Group Donors; Phosphonoacetic Acid; Purine Nucleotides; Pyrazoles; Pyrimidine Nucleotides; Ribonucleosides; Ribose

Quinones are intracellular H2O2 generators that have been used extensively in models of oxidant injury; however, their toxicity is mediated partially through direct conjugation with glutathione (GSH). To focus upon the action of extracellular GSH in preventing H2O2-mediated toxicity, we used 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), which cannot conjugate with GSH but does continuously generate H2O2 through redox cycling. A eukaryotic cell line (3T3-GGT) stably overexpressing gamma-glutamyl transpeptidase (GGT) activity was used to study the role of GGT in utilizing extracellular GSH against DMNQ-induced oxidative stress. DMNQ (0 to 150 microM) caused a dose-dependent decrease of intracellular GSH and adenosine 5'-triphosphate (ATP) in both control and 3T3-GGT cells. The rate of H2O2 escape into the medium during DMNQ exposure was also the same in both cell lines. Administration of GSH helped to maintain intracellular GSH and supported resistance to ATP depletion caused by DMNQ in 3T3-GGT cells but not in control cells. The protective effect of extracellular GSH was completely prevented by acivicin, an inhibitor of GGT. Our results suggest that GGT-dependent breakdown of extracellular GSH for subsequent intracellular resynthesis helped to maintain cellular GSH levels and increased cellular resistance against DMNQ-induced oxidative injury.

Topics: 3T3 Cells; Animals; Blotting, Western; gamma-Glutamyltransferase; Gene Expression; Glutathione; Glycosylation; Humans; Hydrogen Peroxide; Isoxazoles; Mice; Naphthoquinones; Transfection

Acivicin (NSC 163501) and dichloroallyl lawsone (NSC 126771) are potent inhibitors of nucleotide biosynthesis with consequent anti-cancer activity against certain experimental tumors. To determine in detail the metabolic events induced by each inhibitor, we have devised a new two-dimensional chromatographic procedure for measurement of the concentrations of all pyrimidine intermediates and some purine nucleotides from 100 microliter of an extract of cells grown in the presence of [14C]bicarbonate. Addition of acivicin (25 microM) to mouse L1210 leukemia cells causes severe depletion in the cellular levels of CTP and GTP, accumulation of uridine nucleotides, and abrupt but transient increases in the concentrations of the early intermediates of both the pyrimidine and purine pathways. Addition of dichloroallyl lawsone (25 microM) results in a rapid depletion of uridine and cytidine nucleotides; carbamyl aspartate and dihydroorotate accumulate to high levels in an equilibrium ratio of 20.5:1, and orotate, orotidine, and UMP increase transiently before decreasing to levels approaching their original steady states. The predominant inhibitory effects of acivicin are upon the reactions UTP----CTP and XMP----GMP, but there is also an initial transient activation of both the pyrimidine and purine pathways by acivicin. The data obtained with dichloroallyl lawsone are consistent with inhibition of the conversion of UMP----UDP initially followed by potent inhibition of dihydroorotate----orotate.

Topics: Animals; Bicarbonates; Carbon Radioisotopes; Cytidine Triphosphate; Guanosine Triphosphate; Isoxazoles; Kinetics; Leukemia L1210; Mice; Naphthoquinones; Oxazoles; Pyrimidines; Uracil Nucleotides