Possible mechanism of miltefosine-mediated death of Leishmania donovani

Abstract

Miltefosine causes leishmanial death, but the possible mechanism(s) of action is not known. The mode of action of miltefosine was investigated in vitro in Leishmania donovani promastigotes as well as in extra- and intracellular amastigotes. Here, we demonstrate that miltefosine induces apoptosis-like death in L. donovani based on observed phenomena such as nuclear DNA condensation, DNA fragmentation with accompanying ladder formation, and in situ labeling of DNA fragments by the terminal deoxyribonucleotidyltransferase-mediated dUTP-biotin nick end labeling method. Understanding of miltefosine-mediated death will facilitate the design of new therapeutic strategies against Leishmania parasites.

FIG. 1.

Leishmaniacidal activity of miltefosine on L. donovani promastigotes. (A) Cell viability of promastigotes treated with or without different concentrations of miltefosine was measured by MTT assay as described in Materials and Methods. Results are expressed as the means ± SEM (error bars) of three independent experiments. (B) Cell number (live and moving) of promastigotes treated with or without miltefosine (25 μM) at different time points after miltefosine treatment. Results are expressed as the means ± SEM (error bars) of three independent experiments. (C) Promastigotes at different time points after miltefosine (25 μM) treatment.

FIG. 2.

DNA condensation in L. donovani promastigotes after miltefosine treatment. L donovani promastigotes were treated with or without miltefosine (25 μM) for 48 h and were subjected to PI staining. (A) Untreated promastigotes; (B) miltefosine-treated promastigotes. Arrows indicate representative condensed nuclei. Results are representative of three independent experiments.

FIG. 3.

DNA fragmentation in L. donovani promastigotes with or without miltefosine treatment. (A) Genomic DNA (10 μg) from treated and untreated promastigotes was resolved on 1% agarose gel. Lane 1, 1-kb ladder (MBI Fermentas); lane 2, DNA from untreated promastigotes; lane 3, DNA from promastigotes treated with an IC₅₀ dose of miltefosine. (B) In situ TUNEL-stained untreated promastigotes. (C) In situ TUNEL-stained promastigotes treated with an IC₅₀ dose of miltefosine. Arrows indicate representative TUNEL-positive cells. Results are representative of three independent experiments.

FIG. 4.

DNA fragmentation in L. donovani amastigotes with or without miltefosine treatment. (A) Genomic DNA (10 μg) from treated and untreated amastigotes was resolved on 1% agarose gel. Lane 1, 1-kb ladder (MBI Fermentas); lane 2, DNA from untreated amastigotes; lane 3, DNA from amastigotes treated with an IC₅₀ dose of miltefosine. (B) In situ TUNEL-stained untreated amastigotes. (C) In situ TUNEL-stained amastigotes treated with an IC₅₀ dose of miltefosine. Arrows indicate representative TUNEL-positive cells. Results are representative of three independent experiments.

FIG. 5.

In situ analysis (TUNEL staining) of apoptosis in L. donovani amastigote-infected macrophages. (A) Giemsa-stained macrophage infected with amastigotes. (B) Giemsa-stained macrophage infected with amastigotes treated with 25 μM miltefosine. (C) In situ TUNEL-stained untreated amastigote-infected macrophages. (D) In situ TUNEL-stained amastigote-infected macrophages treated with an IC₅₀ dose of miltefosine. Results are representative of three independent experiments.