a Laboratorio de Química Biológica, Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas, Apartado 21827, Caracas 1020A, Venezuela; b Laboratório de Biologia Celular, DUBC, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil
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Abstract |
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Introduction |
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Materials and methods |
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The EP and Y stocks of T. cruzi were used in this study.23 Handling of live T. cruzi was carried out according to established guidelines.24
Drugs and metabolic precursors
MLT was obtained from Sigma Chemical Company (St Louis, MO, USA), while ET-18 and ILM were kindly provided by Dr Simon Croft (London School of Hygiene and Tropical Medicine, UK). Their molecular structures are shown in Figure 1. Ketoconazole was provided by Janssen Pharmaceutica, Caracas, Venezuela. The drugs were added as dimethylsulphoxide (DMSO) solutions; the final DMSO concentration in the culture media never exceeded 1% (v/v) and had no effect by itself on the proliferation of the parasites or Vero cells. [Methyl-14C]choline chloride (55 mCi/mmol) and L-[methyl-14C]methionine (57 mCi/ mmol) were purchased from Amersham International, plc, UK.
In vitro studies
The epimastigote form of the parasite was cultivated in liver infusion tryptose medium,23 supplemented with 10% newborn calf serum (Gibco-BRL) at 28°C with strong agitation (120 rpm). The cultures were initiated with a cell density of 2 x 106 epimastigotes per mL and the drugs were added at a cell density of 0.51 x 107 epimastigotes per mL. Cell densities were measured with an electronic particle counter (model ZBI; Coulter Electronics Inc., Hielah, FL, USA) and by direct counting with a haemocytometer. Cell viability was followed by Trypan blue exclusion using light microscopy. Amastigotes were cultured in Vero cells maintained in minimal essential medium supplemented with 1% fetal calf serum in a humidified 95% air5% CO2 atmosphere at 37°C as described previously.25 Briefly, the cells were infected with 10 tissue culture-derived trypomastigotes per cell for 2 h and then washed three times with phosphate-buffered saline (PBS) to remove non-adherent parasites. Fresh medium with or without drugs was added and the cells were incubated for 96 h with a medium change at 48 h. Quantification of the number of infected cells, the number of parasites per cell by use of light microscopy and statistical analysis of the results was carried out as described previously.25
Studies of lipid composition and biosynthesis
For the analysis of the effects of drugs on the lipid composition of the epimastigotes, total lipids from control and drug-treated cells were extracted and fractionated into neutral and polar lipid fractions by silicic acid column chromatography and gasliquid chromatography (GLC).25 The neutral lipid fractions were first analysed by thin layer chromatography [TLC; on Merck 5721 silica gel plates with heptane:isopropyl ether:glacial acetic acid (60:40:4) as developing solvent] and conventional GLC (isothermal separation in a 4 m glass column packed with 3% OV-1 on Chromosorb 100/200 mesh, with nitrogen as the carrier gas at 24 mL/min and flame ionization detection in a Varian 3700 gas chromatograph). For quantitative analysis and structural assignments the neutral lipids were separated in a capillary high resolution column [25 m x 0.20 mm (internal diameter) Ultra-2 column, 5% phenyl-methyl-siloxane, 0.33 µm film thickness] in a Hewlett-Packard 5890 series II gas chromatograph equipped with an HP5971A mass sensitive detector. The lipids were injected in ethyl-acetate and the column was kept at 50°C for 1 min, then the temperature was increased to 270°C at a rate of 25°C/min and finally to 300°C at a rate of 1°C/min. The carrier gas (helium) flow was kept constant at 1.0 mL/min. The injector temperature was 250°C and the detector was kept at 280°C.
The polar lipid fraction (containing mostly phospholipids) was analysed as described previously;26 briefly, the lipid fractions eluted from the silicic acid column with chloroform:methanol 1:1 (v/v) were pooled and fractionated further by TLC on Merck 5721 silica gel plates using chloroform:methanol:32.5% ammonia w/v (17:7:1 by volume) as developing solvent.27 The phospholipid spots were visualized using iodine, scraped and the total organic phosphorous was measured using the method of Ames & Dubin.28 For the analysis of the fatty acids esterified to the phospholipid fraction, total phospholipids dissolved in 100 µL of chloroform were transmethylated by adding 200 µL of 2% H2SO4 in methanol and incubating at 60°C for 1 h. The reaction was stopped by adding 200 µL of distilled water. The methyl ester was extracted with petroleum ether, dried and quantitatively analysed by GLC in a 2 m x 2 mm (i.d.) column packed with 10% SILAR GT on Chromosorb W (100200 mesh) on a Varian 3700 gas chromatograph with a flame ionization detector. The temperature programme was 150°C for 10 min, followed by a linear temperature increment of 3°C/min up to 205°C, and then isothermal maintainance at this temperature for an additional 25 min; nitrogen gas was used as a carrier at 8 mL/min.26
For the study of de novo synthesis of phospholipids, 0.05 µCi/mL of [methyl-14C]choline chloride or L-[methyl-14C]methionine (55 and 57 mCi/mmol, respectively; Amersham International) was added to the cultures 24 h after the addition of the drug or carrier, and incubation continued for a further 1248 h. The lipids were then extracted, purified and analysed as described above. The radioactive fractions from the TLC were scraped off and counted by liquid scintillation spectrometry in an LKB Rack-Beta counter, working at 80% efficiency for 14C.
Synergy calculations
Synergy is defined in this paper, as in previous publications2933 as an effect produced by a combination of components that is greater than the sum of the effects produced by the components alone. Classical isobolograms were constructed by plotting concentrations of drugs that either alone or in combination induced 50% inhibition of growth (IC50) of epimastigotes after 120 h. Fractional inhibitory concentrations (FICs) were calculated according to Hallander et al.34 and, briefly, are defined as:
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Results |
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The anti-proliferative effects of three LPAs against the epimastigote stage of T. cruzi, equivalent to that present in the Reduviid vector, grown in axenic LIT medium at 28°C are shown in Figures 24. Indistinguishable results were obtained with the Y or EP strain. A clear dose-dependent effect on proliferation can be observed: IC50s after 120 h for ET-18, MLT and ILM were 3, 1 and 3 µM, respectively. At higher concentrations, immediate lytic effects were observed, probably associated with a detergent effect, in accordance with the amphipathic nature of these compounds and their expected high critical micellar concentrations. Against the clinically relevant intracellular amastigote form, grown inside cultured Vero cells at 37°C, LPAs were significantly more potent than against epimastigotes. We show in Table I
that the minimal concentration of the drugs required for complete eradication of the parasites from infected Vero cells were in the range of 0.11 µM. Some effects were also observed on the host cells, basically as slower proliferation rates. Taken together, the results showed that MLT had the most potent anti-parasitic action and lowest toxicity to hosts cells. The effects of all three LPAs on parasite cells were irreversible, as removal of the drug from the growth medium did not lead to resumption of parasite's growth.
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LPAs are phospholipid analogues and it has been reported previously that, among their biochemical effects on tumour cells, alteration of phospholipid synthesis was one of the most prominent.1921 Based on these facts we set out to investigate the effect of these compounds on T. cruzi lipid composition and de novo synthesis, using the epimastigote form as a model. Table II shows the effect of LPAs on the phospholipid composition of these cells after 120 h incubation. The basal composition of control cells (not treated with LPAs) coincides with that reported previously by us and others,26,35,36 PC being by far the most abundant class, followed by phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI), while sphingomyelin constituted a minor component. Incubation with all three LPAs at their IC50 for 120 h induced a uniform reversal of the PC/PE ratio, from 1.5 in control cells to c. 0.67 in those treated with the phospholipid analogues. A significant increase (c. 50%) in the content of PS was also observed in treated cells. On the other hand, Table III
shows that there was no effect of LPAs on the total phospholipid content of epimastigotes. We also investigated effects of LPAs on the fatty acid composition of the phospholipid fraction of T. cruzi epimastigotes. Table IV
shows that incubation of epimastigotes with these compounds was associated with a marked reduction of palmitic acid residues (16:0) and a concomitant increment of stearic (18:0) and oleic (18:2) acid substituents, which accounted for a modest decrease in the saturated to unsaturated fatty acid ratio (SFA/UFA) in treated cells.
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Effects of LPAs on sterol composition of T. cruzi epimastigotes
We also investigated the effects of LPAs on the free sterol composition of epimastigotes using high-resolution GLC coupled to mass spectrometry. It can be seen in Table V that in cells treated with the IC50 of LPAs, the main free sterols of control cells, ergosterol (24-methyl-cholesta-5,7,22-trien- 3ß-ol) and its 24-ethyl analogue, were replaced by its
22- saturated analogues. This indicated inhibition of sterol C-22 desaturase, most probably a secondary effect resulting from the altered phospholipid composition (Table II
).
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Previous observations form our laboratory have shown that a secondary effect of sterol biosynthesis inhibitors is a reduction in PC content associated with an indirect inhibition of PE-PC-N-methyltransferase.26 These findings combined with our present observations that together with their potent inhibitory effects on PC biosynthesis, LPAs also induce a significant modification of the parasite's sterol composition, led us to investigate possible anti-proliferative synergy between the two type of compounds. We carried out these studies using a chequerboard method2933 for combinations of ET-18 and ketoconazole (a sterol C14 demethylase inhibitor), and the results are presented in Figure 6
. A concave isobologram with an FIC index37 of 0.4 clearly indicated synergic effects.
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Discussion |
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The incorporation of L-[methyl-14C]methionine, but not [methyl-14C]choline, into PC clearly showed that the Greenberg's (transmethylation) pathway was the predominant biosynthetic route for PC in this parasite, as seen in fungi and yeasts.38,39 Based on this interpretation we inferred that the blockade of the incorporation of radioactivity from L-[methyl-14C]methionine into PC by ET-18 was most probably due to direct inhibition of PE-PC-N-methyltransferase, an explanation consistent with the increase in PS levels seen in treated cells (Table II). As the antiproliferative and biochemical effects of MLT and ILM were essentially identical to those observed with ET-18, it is reasonable to assume that their mechanisms of action are also similar. LPAs have also been shown to inhibit PC biosynthesis in mammalian cells, in which the CDP-choline or Kennedy's pathway is predominant,19,20,38 probably acting at the level of cytidyltransferase.19 However, the IC50s of ET-18 and MLT needed to elicit these effects in canine kidney cells were 10- to 20-fold higher that those found for T. cruzi epimastigotes in the present study.19,20 Thus, a specific inhibition of the Greenberg's pathway in T. cruzi by LPAs at low micromolar levels could account for their selective anti-parasitic activity.
We also observed in previous studies an inversion of the PC/PE ratio in cells treated with sterol biosynthesis inhibitors,26 but concluded that the effect was secondary to the altered sterol composition of the cellular membranes. The synergic effects resulting from the combined action of ET-18 and ketoconazole on the proliferation of T. cruzi epimastigotes (Figure 6) can be explained as both compounds acting on the same biochemical target through different mechanisms. This finding suggests that well- established and widely available antifungal sterol biosynthesis inhibitors could potentiate the in vivo anti-parasitic effects of LPAs. Further in vitro and in vivo studies are currently underway to test this hypothesis.
Conclusions
The results of the present study led us to conclude that a possible explanation for the selective anti-T. cruzi effects of LPAs is a specific inhibition of the parasite's PC biosynthesis, which seems to proceed through the Greenberg's (transmethylation) pathway, in contrast to the CDP-choline pathway used by host cells. We have also found that this anti-proliferative activity can be potentiated by sterol biosynthesis inhibitors and provided a possible molecular explanation for these effects. Although our studies have been centred around T. cruzi, we are currently extending them to related organisms such as Leishmania mexicana and Leishmania braziliensis, which are also susceptible to LPAs in vitro and in vivo.911,13
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Note added in proof |
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Acknowledgments |
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Notes |
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References |
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Received 5 May 2000; returned 27 July 2000; revised 3 October 2000; accepted 20 November 2000