Miltefosine induces apoptosis-like death in Leishmania donovani promastigotes

Abstract

Miltefosine (hexadecylphosphocholine [HePC]) has proved to be a potent oral treatment for human visceral leishmaniasis due to Leishmania donovani. The molecular mechanisms that contribute to the antileishmanial activity of HePC are still unknown. We report that in wild-type promastigotes of Leishmania donovani HePC is able to induce a cell death process with numerous cytoplasmic, nuclear, and membrane features of metazoan apoptosis, including cell shrinkage, DNA fragmentation into oligonucleosome-sized fragments, and phosphatidylserine exposure. None of these changes were detected in an HePC-resistant clone treated with the same drug concentration. Therefore, HePC does not appear to kill L. donovani promastigotes by a direct toxic mechanism but, rather, kills the promastigotes by an indirect one. Pretreatment of wild-type promastigotes with two broad caspase inhibitors, z-Val-Ala-DL-Asp(methoxy)-fluoromethylketone and Boc-Asp(methoxy)-fluoromethylketone, as well as a broad protease inhibitor, calpain inhibitor I, prior to drug exposure interfered with DNA fragmentation but did not prevent cell shrinkage or phosphatidylserine externalization. These data suggest that at least part of the apoptotic machinery operating in wild-type promastigotes involves proteases. Identification of the death-signaling pathways activated in HePC-sensitive parasites appears to be essential for a better understanding of the molecular mechanisms of action and resistance in these parasites.

FIG. 1.

(Top) Chemical structure of HePC; (bottom) cytotoxic activity of HePC on L. donovani promastigotes. The viability of WT (open circles) and HePC-R40 (closed circles) parasites after a 3-day incubation in the presence of graded concentrations of HePC was assessed by the colorimetric MTT assay and is expressed as the percentage of surviving promastigotes compared to the number of surviving untreated cells. Data are the means of five independent experiments performed in triplicate, and standard deviations are represented by error bars.

FIG. 2.

Morphology and cell size of L. donovani WT promastigotes cultured for 24 h in fresh complete medium at 26°C in the absence or presence of different concentrations of HePC. The results of light microscopy (magnification, ×1,000) (A) and flow cytometry analysis (B) of the cell sizes of WT promastigotes either untreated (A1 and B1) or exposed to 10 μM (A2 and B2), 20 μM (A3 and B3), or 40 μM HePC (A4 and B4) are shown.

FIG. 3.

DNA fragmentation analysis by agarose gel electrophoresis. The DNA profiles for untreated or HePC-treated L. donovani promastigotes after 24 h of incubation at 26°C are shown. Lane M, molecular size marker (pb, base pairs). WT promastigotes were untreated (lane 1) or were exposed to 10 μM (lane 2), 20 μM (lane 3), or 40 μM HePC (lane 4); HePC-R40 promastigotes were untreated (lane 5) or were exposed to 20 μM (lane 6), 40 μM (lane 7), or 80 μM HePC (lane 8). The results are representative of those from three independent experiments.

FIG. 4.

Cell size and analysis of DNA content by flow cytometry of untreated or HePC-treated L. donovani promastigotes after 24 h of incubation at 26°C. The light-scattering properties (A) and the DNA fragmentation (B) of WT promastigotes untreated (A1 and B1) or exposed to 40 μM HePC (A2 and B2) and of HePC-R40 promastigotes untreated (A3 and B3) or exposed to 40 μM HePC (A4 and B4) are shown. Cells containing amounts of DNA found in the sub-G₁ peak region (pseudohypodiploid cells) are in region M₁, and their percentages were calculated for each histogram. The data are representative of those from at least six independent experiments. SSC-H, side scatter; FSC-H, forward scatter; FL3-H, fluorescence intensity.

FIG. 5.

DNA fragmentation analysis by flow cytometry. The percentages of pseudohypodiploid cells of either WT promastigotes (top) or HePC-R40 promastigotes (bottom) were determined after 24 h (open bars) or 48 h (closed bars) of incubation at 26°C in the absence or presence of different concentrations of HePC. The data are the means of five independent experiments, and standard deviations are represented by errors bars.

FIG. 6.

Phosphatidylserine exposure analysis by flow cytometry. The percentages of annexin V-FITC-positive WT promastigotes (top) or HePC-R40 promastigotes (bottom) were determined after 24 h (open bars) or 48 h (closed bars) of incubation at 26°C in the absence or presence of 40 μM HePC. The data are the means of three independent experiments, and standard deviations are represented by errors bars.

FIG. 7.

Effects of protease inhibitors on HePC-induced DNA fragmentation in WT promastigotes. Parasites were incubated for 2 h in the presence or absence of the protease inhibitors z-VAD-fmk, Boc-D-fmk, or E64 (100 μM), calpain inhibitor I (20 μM), or lactacystin (50 μM) prior to the addition of HePC (40 μM). Promastigotes were incubated for an additional 24 h at 26°C. The percentage of pseudohypodiploid cells was determined by flow cytometry analysis. The data are the means of three independent experiments, and standard deviations are represented by errors bars.