Dept. de Ultra-estrutura e Biologia Celular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, 21045900, Rio de Janeiro-RJ, Brazil
Received 25 March 2004; returned 19 May 2004; revised 27 May 2004; accepted 29 June 2004
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Abstract |
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Methods: Quantification and ultrastructural analysis of the effect of lysophospholipid analogues on promastigote forms and on infected peritoneal macrophages, and flow cytometry analysis of treated promastigotes labelled with propidium iodide and rhodamine 123 (Rh123).
Results: The lysophospholipid analogues presented potent antiproliferative activity with IC50/3 days of 1.93.4 µM for promastigotes and 4.29.0 µM for intracellular amastigotes. Treatment with these analogues in Schneider medium for 1 day led to a dose-dependent decrease in Rh123 fluorescence, an effect more accentuated in edelfosine-treated parasites, suggesting interference with the potential of the mitochondrial membrane. In both forms of L. amazonensis, edelfosine induced extensive mitochondrial damage, multinucleation and, in promastigotes, also led to plasma membrane alterations, formation of autophagic structures and membranous arrangements inside the flagellar pocket.
Conclusions: The alkylglycerophosphocholines edelfosine and ilmofosine were more active than the alkylphosphocholine miltefosine against promastigotes and intracellular amastigotes of L. amazonensis, and ultrastructural and flow cytometry data indicate the mitochondrion as a target of edelfosine.
Keywords: L. amazonensis , alkylglycerophosphocholines , flow cytometry , chemotherapy , electron microscopy
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Introduction |
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Materials and methods |
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L. amazonensis (strain MHOM/BR/77/LTB0016) amastigote forms were obtained from lesions of infected Swiss mice.13 The parasites were added to Schneider medium supplemented with 10% fetal calf serum (FCS), 100 IU penicillin, 100 mg/mL streptomycin and 1 mM L-glutamine, pH 7.2, at 25°C, leading to differentiation to promastigotes, which were maintained for up to four passages in this same medium.14
Edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) was obtained from Bachem Ltd (St Helens, UK); ilmofosine (1-hexadecylthio-2-methoxymethyl-rac-glycero-3-phosphocholine) and miltefosine (hexadecylphosphocholine) were from Sigma Chemical Co. (St Louis, MO, USA). Stock solutions of the lysophospholipid analogues were prepared in phosphate buffered saline (PBS), pH 7.2.
Effect of the lysophospholipid analogues on the proliferation of L. amazonensis
Culture promastigotes were resuspended in Schneider medium (5 x 106 cells/mL) and plated in 24-well plates at 25°C, in the absence or presence of different concentrations of the analogues. Cell counts were performed up to 3 days of treatment and the corresponding IC50 value was calculated. This parameter expresses the concentration of each analogue that leads to 50% inhibition of parasite proliferation, and was obtained from at least three experiments and expressed as mean ± S.D. After 1 day of treatment with edelfosine, the parasites were processed for transmission and scanning electron microscopy (TEM and SEM, respectively).
Effect of the lysophospholipid analogues on L. amazonensis-infected macrophages
Mouse peritoneal macrophages were infected with promastigotes at a ratio of 10:1 parasites/host cell. After interaction for 1 h, non-internalized parasites were removed by washing with Dulbecco's modified Eagle's medium plus 10% FCS. Fresh medium, with or without the analogues, was added to the cultures and changed every 2 days. After 13 days of treatment, the cultures were fixed, stained with Giemsa and the percentage of infection and the number of intracellular parasites quantified. Alternatively, the cultures were processed for TEM.
Ultrastructural analysis
Promastigotes or infected cell cultures treated with edelfosine and the corresponding controls were washed with PBS and processed for electron microscopy. For SEM, the parasites were adhered to poly-L-lysine-coated coverslips, fixed with 2.5% glutaraldehyde in 0.1 M Na-cacodylate buffer (pH 7.2) at room temperature for 40 min and post-fixed with 1% OsO4 in the same buffer for 30 min. The cells were dehydrated in an ascending acetone series, dried by the critical point method with CO2, mounted on silver Cellotape on aluminium stubs, coated with a 20 nm thick layer of gold and examined in a 940 DSM Zeiss microscope (Oberkochen, Germany). For TEM, after washing in PBS, the parasites or macrophage cultures were fixed with 2.5% glutaraldehyde and 2.5 mM CaCl2 in a cacodylate buffer for 1 h at 4°C, post-fixed in 1% OsO4, 0.8% potassium ferricyanide and 2.5 mM CaCl2 in a 0.1 M cacodylate buffer for 1 h, dehydrated as described above and embedded in Polybed 812 resin. Ultrathin sections (Leica Ultracuts, UCT, Vienna, Austria) were stained with uranyl acetate and lead citrate and then examined in an EM10C Zeiss microscope.
Flow cytometry analysis
Promastigotes (5 x 106 cells/mL) were treated with the lysophospholipid analogues at 25°C for 10 min in PBS or else for 13 days in Schneider medium. Thereafter, 500 µL aliquots of the cultures were added to the same volume of a solution containing 20 µg/mL propidium iodide (PI) plus 12 µg/mL rhodamine 123 (Rh123) and then incubated at 25°C for 20 min. The material was kept on ice until analysis. Data acquisition and analysis were performed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with Cell Quest software (Joseph Trotter, Scripps Research Institute, San Diego, CA, USA). A total of 10 000 events were acquired in the region previously established as that corresponding to the parasites. Alterations in the fluorescence for Rh123 were quantified using an index of variation (IV) obtained by the equation (MTMC)/MC, where MT is the median of fluorescence for treated parasites and MC that for control parasites. Negative IV values correspond to a depolarization of the mitochondrial membrane.
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Results |
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Discussion |
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In promastigotes of L. amazonensis, edelfosine induced alterations at the plasma membrane and formation of autophagic structures, which could have been the result of recycling of abnormal membranes during the processing of damaged organelles, as described in suramin-treated Trypanosoma rhodesiense.15 Blebbing and ruffling of the plasma membrane have been described in Trypanosoma cruzi16 and HL-60 leukaemic cells17 after treatment with edelfosine, in similar experimental conditions.
Since ultrastructural analysis demonstrated that treatment with lysophospholipid analogues induced alterations at the membrane and mitochondrion of promastigotes, we incubated treated parasites with PI and Rh123. Treatment with edelfosine at concentrations above 1 µM in PBS for 10 min led to a gradual increase in membrane permeabilization, whereas the addition of 10% FCS prevented such an effect, even at 40-fold higher concentrations. This protective effect of serum has been reported in experiments with tumour cells18,19 and T. cruzi16 and is associated with a decrease in free analogue, due to its interaction with serum components. The decrease in Rh123 fluorescence after treatment of promastigotes in Schneider medium for 1 day with the analogues, especially edelfosine suggests interference with the hydrogenionic potential of the mitochondrial membrane, as reported in paromomycin-treated L. donovani.20 The reduced retention of Rh123 is not due to secondary plasma membrane permeabilization; since in our experiments the percentage of PI-labelled parasites after treatment was similar to that of untreated ones. These results are in agreement with the extensive damage of the parasite mitochondrion, as detected by TEM.
The multinucleation observed in promastigotes and amastigotes of L. amazonensis has been reported in mammalian tumour lineages treated with edelfosine, in which supplementation with phosphatidylcholine precursors partially reverted the observed polyploidy induced by blockage of cytokinesis.2123 The mitochondrial damage, with membranous arrangements inside this organelle and in the flagellar pocket, and the formation of autophagic structures observed in edelfosine-treated L. amazonensis have also been described when this parasite or T. cruzi were treated with inhibitors of sterol synthesis.2427 Such alterations are associated with depletion of ergosterol and alteration of the physical properties of the membranes. We have previously shown that lysophospholipid analogues inhibit the proliferation and the synthesis of phospholipids and sterols in T. cruzi.16,28 Further experiments on lipid characterization and quantification of treated L. amazonensis are needed in order to ascertain if a similar mode of action is also operative in this trypanosomatid, with the morphological alterations in membranous systems linked with interference in the biosynthesis of lipids.
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Acknowledgements |
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Footnotes |
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References |
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