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

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-18OCH(3), miltefosine and ilmofosine were 3, 1 and 3 muM, 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 muM for ET-18-OCH3, miltefosine and ilmofosine, respectively. Analysis of the lipid composition of epimastigotes exposed to LPAs at their IC50 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-C-14]methionine into PC, but not from [methyl-C-14]choline. ET-18-OCH3 inhibited the incorporation of L-[methyl-14C]methionine into PC with an IC50 of 2 muM, 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-OCH3 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.