Apoptosis-like cell death in Leishmania donovani treated with KalsomeTM10, a new liposomal amphotericin B

The present study aimed to elucidate the cell death mechanism in Leishmania donovani upon treatment with KalsomeTM10, a new liposomal amphotericin B. Methodology/Principal findings We studied morphological alterations in promastigotes through phase contrast and scanning electron microscopy. Phosphatidylserine (PS) exposure, loss of mitochondrial membrane potential and disruption of mitochondrial integrity was determined by flow cytometry using annexinV-FITC, JC-1 and mitotraker, respectively. For analysing oxidative stress, generation of H2O2 (bioluminescence kit) and mitochondrial superoxide O2 − (mitosox) were measured. DNA fragmentation was evaluated using terminal deoxyribonucleotidyl transferase mediated dUTP nick-end labelling (TUNEL) and DNA laddering assay. We found that KalsomeTM10 is more effective then Ambisome against the promastigote as well as intracellular amastigote forms. The mechanistic study showed that KalsomeTM10 induced several morphological alterations in promastigotes typical of apoptosis. KalsomeTM10 treatment showed a dose- and time-dependent exposure of PS in promastigotes. Further,study on mitochondrial pathway revealed loss of mitochondrial membrane potential as well as disruption in mitochondrial integrity with depletion of intracellular pool of ATP. KalsomeTM10 treated promastigotes showed increased ROS production, diminished GSH levels and increased caspase-like activity. DNA fragmentation and cell cycle arrest was observed in KalsomeTM10 treated promastigotes. Apoptotic DNA fragmentation was also observed in KalsomeTM10 treated intracellular amastigotes. KalsomeTM10 induced generation of ROS and nitric oxide leads to the killing of the intracellular parasites. Moreover, endocytosis is indispensable for KalsomeTM10 mediated anti-leishmanial effect in host macrophag

Analysis of genomic DNA fragmentation in KalsomeTM10, Ambisome and amphotericin B treated intracellular amastigotes analysed through TUNEL assay.

<p><i>L</i>. <i>donovani</i> infected murine peritoneal macrophages were either left untreated (a) or treated with KalsomeTM10 (b) at 0.5 μg/ml, Ambisome (c) at 1.0 μg/ml and amphotericin B (d) at 2.0 μg/ml. After 48 h, infected macrophages were stained using TUNEL and analysed by fluorescence microscopy. Total nuclei are visualized in red (PI) and TUNEL-positive nuclei are stained in green (FITC). The images are representative of three independent experiments.</p

PS externalization in KalsomeTM10 treated promastigotes.

<p>Promastigotes, untreated (a and b) and treated with 2.5 μg/ml (c) and 5.0 μg/ml (d) of KalsomeTM10 for 1 h, and treated with 2.5 μg/ml (e) and 5.0 μg/ml (f) of KalsomeTM10 for 2 h, were co stained with annexin V-FITC and PI, and analysed by flow cytometry. The dot plots are representative of one of the three independent experiments. (g) Bar graphs representing mean % PS⁺/PI⁻ cells.<i>**P</i><0.001.</p

Generation of lipid peroxidation products on treatment with KalsomeTM10.

<p>Promastigotes were treated with different concentrations of kalsomeTM10 for 2 h in the absence (a) and presence of NAC (b), and lipid peroxidation products were determined flourometrically. Results are presented as means + SD; n = 3. <i>P</i><0.05.</p

ROS and mitochondrial super-oxide production, and depletion of GSH pool in KalsomeTM10 treated promastigotes.

<p>Promastigotes incubated at different time points with KalsomeTM10 at 2.5 μg/ml (a i) and 5.0 μg/ml (a ii), with or without NAC (1 mM and 10 mM). Cellular ROS was measured by bioluminescence assay. Results are presented as means + SD; n = 3. Promastigotes, untreated (b i and ii) and treated with 2.5 μg/ml of KalsomeTM10 for 30 min (b iii) and 1 h (b iv), and treated with 5.0 μg/ml of KalsomeTM10 for 30 min (b v) and 1 h (b vi), were stained with mitosox, and analysed by flow cytometry. The histograms are representative of three independent experiments. (c) Bar graphs representing mean % superoxide⁺ cells. (d) Promastigotes treated with 2.5 μg/ml of KalsomeTM10 at different time points and GSH concentration determined flourometrically. Results are presented as means + SD; n = 3. <i>*P</i><0.01, <i>**P</i><0.001.</p

Cellular morphology of KalsomeTM10 treated <i>L</i>. <i>donovani</i> promastigotes.

<p>Phase contrast micrographs of promastigotes after exposure to KalsomeTM10 for 4 h. Untreated control (a), promastigotes exposed to 1.0 μg/ml (b), 2.5 μg/ml (c) and 5.0 μg/ml (d). Images are representative of three independent experiments.</p

Scanning electron microscopy of promastigotes treated with KalsomeTM10.

<p>Promastigotes were either left untreated (a) or treated with 5.0 μg/ml of Kalsome TM10 (b) for 2 h and then analysed for surface topology. SEM micrographs show rounded and distorted shape in KalsomeTM10 treated promastigotes compared to controls. Scale bars: 5 μM. Images are representative of three independent experiments.</p

Role of ROS and nitric oxide generation on in-vitro leishmanicidal effect of KalsomeTM10 on intracelluar amsastigotes.

<p>Macrophages, infected with promastigotes, for 24 h were pretreated with ROS scavangers like NAC (10 mM), PEG-Cat (500 units) and PEG-SOD (500 units) and nitric oxide inhibitor L-NMMA (2 mM) for 30 mins and then treated with KalsomeTM10 (0.5 μg/ml) for 1h. After washing, incubation for 48 h post drug treatment agan in the prescence of these scavangers and inhibitor was carried out and then the infected macrophages were geimsa stained and counted on a light microscope to determine infectivity index (Number of amastigotes per 100 macrophages) that was represented as bar graph. Results are presented as means + SD; n = 2.<i>***P</i><0.0001.</p

Analysis of cell cycle arrest in <i>L</i>. <i>donovani</i> promastigotes treated with KalsomeTM10.

<p>Promastigotes, either left untreated (a i) or treated with 2.5 μg/ml (a ii) and 5.0 μg/ml (a iii) of kalsomeTM10 for 2 h and untreated (b i) or treated with 2.5 μg/ml (b ii) and 5.0 μg/ml (b iii) of KalsomeTM10 for 4 h, were analysed for cell cycle through flow cytometry and plotted as histograms which are representative of three independent experiments. (c) Mean % Sub G0 cells represented as bar graphs.<i>**P</i><0.001, <i>***P</i><0.0001.</p

In-vitro leishmanicidal effect of KalsomeTM10 on peritoneal macrophages infected with promastigotes of <i>L</i>. <i>donovani</i>.

<p>Macrophages, infected with promastigotes, were treated with different concentrations of KalsomeTM10 for 72 h post infection. The infected macrophages on coverslips were geimsa stained and counted on a light microscope (a). Number of amastigotes per 100 macrophages was represented as bar graph. Results are presented as means + SD; n = 3. Infected macrophages untreated (b i) and treated with 10 ng/ml (b ii), 50 ng/ml (b iii), 100 ng/ml (b iv), 500 ng/ml (b v) and 1000 ng/ml (b vi) of KalsomeTM10, were giemsa stained and imaged on a light microscope. Pictorial representation of three independent experiments is depicted.</p