title: Mechanism of action of anti-proliferative lysophospholipid analogues against the protozoan parasite Trypanosoma cruzi: potentiation of in vitro activity by the sterol biosynthesis inhibitor ketoconazole.
abstract: We investigated the mechanism of action of metabolically stable lysophospholipid analogues (LPAs), with potent anti-tumour and anti-protozoal activity against Trypanosoma cruzi, the causative agent of Chagas' disease. Against the axenically grown epimastigote form of the parasite, the IC(50)s after 120 h for ET-18-OCH(3), miltefosine and ilmofosine were 3, 1 and 3 microM, respectively; at higher concentrations immediate lytic effects were observed. Eradication of the intracellular amastigote, grown inside Vero cells, was achieved at 0.1, 0.1 and 1 microM for ET-18-OCH(3), miltefosine and ilmofosine, respectively. Analysis of the lipid composition of epimastigotes exposed to LPAs at their IC(50) for 120 h showed that the ratio of phosphatidyl-choline (PC) to phosphatidylethanolamine (PE) changed from 1.5 in control cells to c. 0.67 in those treated with the analogues. A significant increase in the content of phosphatidylserine was also observed in treated cells. Intact epimastigotes efficiently incorporated radioactivity from L-[methyl-(14)C]methionine into PC, but not from [methyl-(14)C]choline. ET-18-OCH(3) inhibited the incorporation of L-[methyl-(14)C]methionine into PC with an IC(50) of 2 microM, suggesting that inhibition of the de novo synthesis through the Greenberg's pathway was a primary effect underlying the selective anti-parasitic activity of this compound. Antiproliferative synergism was observed as a consequence of combined treatment of epimastigotes with ET-18-OCH(3) and ketoconazole, a sterol biosynthesis inhibitor, probably due to the fact that a secondary effect of the latter is also a blockade of PC synthesis at the level of PE-PC-N-methyl-transferase.
authors.affiliation: Laboratorio de Química Biológica, Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas, Apartado 21827, Caracas 1020A, Venezuela.
authors.forename: R
authors.initials: R
authors.lastname: Lira
authors.forename: L M
authors.initials: LM
authors.lastname: Contreras
authors.forename: R M
authors.initials: RM
authors.lastname: Rita
authors.forename: J A
authors.initials: JA
authors.lastname: Urbina
mesh_terms: D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; D004357:Drug Synergism; D006801:Humans; D007654:Ketoconazole / Q000494:pharmacology*; D008246:Lysophospholipids / Q000494:pharmacology*; D021261:Parasitic Sensitivity Tests; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D013261:Sterols / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000737:chemistry; D014349:Trypanosoma cruzi / Q000187:drug effects* / Q000378:metabolism
chemical_list: D000935:Antifungal Agents; D000981:Antiprotozoal Agents; D008246:Lysophospholipids; D010743:Phospholipids; D013261:Sterols; D007654:Ketoconazole