Dicoumarol impairs mitochondrial electron transport and pyrimidine biosynthesis in human myeloid leukemia HL-60 cells.
Dicoumarol, a competitive inhibitor of NAD(P)H:quinone oxidoreductase 1 (NQO1), increases intracellular superoxide and affects cell growth of tumor cells. This work was set to establish a mechanistic link between dicoumarol, superoxide and cell cycle alterations in HL-60 cells. Using ES936, a mechanism-based irreversible inhibitor of NQO1, we demonstrate that NQO1 inhibition is not a major factor involved in superoxide boost. Mitochondrial Complexes II, III and IV were directly inhibited by dicoumarol. Succinate, which inhibits superoxide generation by reversed electron flow in Complex II, significantly decreased superoxide boost in dicoumarol-treated cells and in isolated mitochondria incubated with dicoumarol and decylubiquinol. Superoxide generation in cells was strongly potentiated by blocking the quinone site of Complex II with thenoyltrifluoroacetone, supporting the involvement of cytochrome b560 to drive electrons for increasing superoxide. Simultaneous inhibition of the mitochondrial chain upstream ubiquinone and displacement of succinate from the Complex II active site is proposed as a major mechanism to explain how dicoumarol increases superoxide in HL-60 cells. Dicoumarol-treated cells accumulated in S phase due to the impairment of pyrimidine biosynthesis at dihydroorotate dehydrogenase step because blockade was overcome by addition of exogenous uridine or orotate, but not by dihydroorotate. We demonstrate for the first time that dicoumarol inhibits mitochondrial electron transport, induces superoxide release by reversed electron flow in Complex II, and inhibits pyrimidines biosynthesis. These actions must be taken into account when considering dicoumarol effects on cells.
González-Aragón
David
D
Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Ciencias, Universidad de Córdoba, Campus Rabanales, Edificio Severo Ochoa, 3(a) planta, 14014 Córdoba, Spain.
Ariza
Julia
J
Villalba
José M
JM
D001728:Dicumarol / Q000494:pharmacology*; D004579:Electron Transport / Q000187:drug effects; D042963:Electron Transport Complex II / Q000037:antagonists & inhibitors / Q000502:physiology; D014450:Electron Transport Complex III / Q000037:antagonists & inhibitors; D003576:Electron Transport Complex IV / Q000037:antagonists & inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D018922:HL-60 Cells; D006801:Humans; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000037:antagonists & inhibitors*; D011743:Pyrimidines / Q000096:biosynthesis*; D016196:S Phase / Q000187:drug effects; D013481:Superoxides / Q000378:metabolism; D013804:Thenoyltrifluoroacetone / Q000494:pharmacology
D004791:Enzyme Inhibitors; D011743:Pyrimidines; D013481:Superoxides; D013804:Thenoyltrifluoroacetone; D001728:Dicumarol; D042963:Electron Transport Complex II; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human; D003576:Electron Transport Complex IV; D014450:Electron Transport Complex III; C030986:pyrimidine