Miltefosine-induced apoptotic cell death on Leishmania major and L. tropica strains

Abstract

The aim of this study was to assess the cytotoxic effects of various concentrations of miltefosine on Leishmania major (MRHO/IR/75/ER) and L. tropica (MHOM/IR/02/Mash10) promastigotes and to observe the programmed cell death features. The colorimetric MTT assay was used to find L. major and L. tropica viability and the obtained results were expressed as 50% inhibitory concentration (IC50). Also, 50% effective doses (ED50) for L. major and L. tropica amastigotes were also determined. Annexin-V FLUOS staining was performed to study the cell death properties of miltefosine using FACS analysis. Qualitative analysis of the total genomic DNA fragmentation was performed by agarose gel electrophoresis. Furthermore, to observe changes in cell morphology, promastigotes were examined using light microscopy. In both strains of L. major and L. tropica, miltefosine induced dose-dependent death with features of apoptosis, including cell shrinkage, DNA laddering, and externalization of phosphatidylserine. The IC50 was achieved at 22 µM and 11 µM for L. major and L. tropica after 48 hr of incubation, respectively. ED50 of L. major and L. tropica amastigotes were 5.7 µM and 4.2 µM, respectively. Our results indicate that miltefosine induces apoptosis of the causative agent of cutaneous leishmaniasis in a dose-dependent manner. Interestingly, L. major did not display any apoptotic changes when it was exposed to miltefosine in concentrations sufficient to kill L. tropica.

Keywords: ED50; IC50; Leishmania major; Leishmania tropica; apoptosis; miltefosine.

Fig. 1

The viability of L. major and L. tropica promastigotes, in the presence of various concentrations of HePC, assessed by MTT. Each point represents the means of 3 independent tests. Dotted line: L. tropica promastigotes, solid line: L. major.

Fig. 2

Number of promastigotes treated with or without miltefosine at different time points after 48 hr. Dotted line: L. major (MRHO/IR/75/ER) promastigotes and L. tropica (MHOM/IR/02/Mash10) control group, solid line: L. tropica and L. major-treated cells.

Fig. 3

Analysis of morphology in light microscopy (magnification, ×100) and flow cytometry analysis of L. major and L. tropica promastigotes following treatment with 22 µM and 11 µM HePC, respectively, at different time points after miltefosine treatment. (A) L. major 24 hr after treatment. (B) L. major 48 hr after treatment. (C) L. tropica 24 hr after treatment. (D) L. tropica 48 hr after treatment. Lower right region (LR) belongs to apoptotic cells (annexin positive) and upper left region (UL) belongs to necrotic cells (PI positive).

Fig. 4

DNA fragmentation detected with agarose gel electrophoresis of L. major (right) and L. tropica (left). C, Not treated; Lanes 12, 24, 36, and 48 hr incubation periods after treatment with miltefosine. Lane M, size marker.