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Aloe vera leaf exudate induces a caspase-independent cell death in Leishmania donovani promastigotes

Avijit Dutta^1,, Suman Bandyopadhyay^1,, Chitra Mandal¹ and Mitali Chatterjee²

¹ Department of Immunobiology, Indian Institute of Chemical Biology, 4 Raja SC Mullick Road, Kolkata 700 032, India

² Department of Pharmacology, Institute of Postgraduate Medical Education & Research, 244 B Acharya JC Bose Road, Kolkata 700 020, India

Correspondence
Chitra Mandal
cmandal{at}iicb.res.in
Mitali Chatterjee
ilatim{at}vsnl.net

Received 30 October 2006
Accepted 12 December 2006

Abbreviations: AVL, Aloe vera leaf exudate; H₂DCFDA, 2',7'-dichlorodihydrofluorescein diacetate; MFI, mean fluorescence intensity; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PCD, programmed cell death; PI, propidium iodide; ROS, reactive oxygen species; TUNEL, terminal deoxynucleotidyltransferase mediated dUTP end labelling.

Present address: Dept of Pharmacology, Institute of Postgraduate Medical Education & Research, 244 B Acharya JC Bose Road, Kolkata 700 020, India.

Present address: Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-1601, USA.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Protozoan parasites of the genus Leishmania are obligately intracellular, reside in mononuclear phagocytes and cause a wide spectrum of clinical manifestations resulting in substantial morbidity and mortality in an estimated 12 million people worldwide (Desjeux, 2004). The major clinical presentations depend upon the causative species and immunological state of the host. These range from a simple cutaneous lesion through to the disfiguring mucocutaneous leishmaniasis and finally to the visceralized form or kala-azar, which is fatal if left untreated (Murray et al., 2005). In recent years, an unprecedented increase in unresponsiveness to sodium antimony gluconate, the first line of treatment, in visceral leishmaniasis patients has been observed in the Indian subcontinent, a major endemic area of visceral leishmaniasis (Sundar & Chatterjee, 2006).

In the ongoing search for better leishmanicidal compounds (Dupouy-Camet, 2004; Handman, 2001), plant-derived products (Kayser et al., 2003; Akendengue et al., 1999) are being evaluated. We have recently reported the efficacy of the Aloe vera leaf exudate (AVL) against promastigotes of Leishmania braziliensis, Leishmania mexicana, Leishmania tropica, Leishmania major and Leishmania infantum (Dutta et al., 2007). Among various mechanisms for mediating parasiticidal activity, programmed cell death (PCD) appears to be the most preferred, as has been observed in kinetoplastids (Szallies et al., 2002; Arnoult et al., 2002) in response to diverse stimuli, e.g. heat shock (Moreira et al., 1996), chemotherapeutic agents such as pentostam, amphotericin B (Lee et al., 2002), oxidants such as H₂O₂ (Das et al., 2001) or even serum depletion (Zangger et al., 2002). In this study, our objective was to evaluate the putative mechanism(s) mediating the leishmanicidal property of AVL. Herein, we report that AVL induces PCD in L. donovani promastigotes via a protease- and caspase-independent signalling pathway involving changes in the mitochondrial membrane potential and release of cytochrome c from hypopolarized mitochondria and culminating in DNA fragmentation.

	METHODS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Materials. M-199 medium and fetal calf serum (FCS) were obtained from Gibco-BRL, DMSO from SRL, methanol from Merck, Apoptotic DNA ladder kit from Roche, 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA), Hoechst 33258, 1,4-diazabicyclo[2.2.2]octane, JC-1, propidium iodide (PI), mitotracker deep red and fluo-3/AM from Molecular Probes, and cytofix, cytoperm, murine anti-cytochrome c antibody, anti-murine IgG–FITC, annexin V–FITC and Apo-Direct kit from BD Biosciences. All other chemicals were from Sigma unless stated.

Parasite culture. Promastigotes of L. donovani (MHOM/IN/83/AG83) were routinely cultured at 22 °C in M-199 medium supplemented with 10 % heat-inactivated FCS and gentamicin (100 µg ml^–1) and subcultured every 72 h.

Preparation of AVL. Aloe vera syn. Aloe berbadensis Mill. was collected from Midnapore, West Bengal, and a voucher specimen was obtained from the Botanical Survey of India, Shibpore, Howrah. Initially, fresh succulent leaves of Aloe vera (Aloe berbadensis Mill.) were collected and crushed in an electric grinder and this was considered the crude extract. Subsequently, the inner gelatinous portion and the outer leafy coat were manually separated and similarly crushed. All three components, i.e. crude, gel and leaf, were individually tested for their antileishmanial activity. As the maximal activity was identified in the exudate of the leaf (unpublished), it was lyophilized and stored at 4 °C; in all experiments, the DMSO (Sigma) soluble fraction was used (3.5 mg ml^–1). This fraction is referred to as leafy exudate of Aloe vera (AVL).

In vitro cytotoxicity assay. Exponential-phase promastigotes were resuspended in modified RPMI 1640 medium (phenol red free) supplemented with 10 % FCS and 100 µg gentamicin ml^–1 (Medium A); parasites were seeded in 96-well tissue culture plates (5x10⁴ in 250 µl per well) and exposed to increasing concentrations of AVL (0–300 µg ml^–1) for 72 h at 22 °C. Parasite viability was evaluated using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Dutta et al., 2005) wherein the amount of formazan produced is directly proportional to the number of metabolically active cells. Accordingly, the A₄₉₂ represented the number of live cells and the IC₅₀, i.e. the concentration that decreased cell growth by 50 %, was determined by graphical extrapolation.

Flow cytometric analysis of externalized phosphatidylserine in L. donovani promastigotes. Double staining with annexin V–FITC and PI was performed as previously described (Mehta & Shaha, 2004). Briefly, promastigotes were incubated with AVL (87.5 µg ml^–1, 24 h); cells were centrifuged (3500 r.p.m. x 10 min), washed twice in PBS (0.02 M, pH 7.2) and resuspended in annexin-V binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂; pH 7.4). Annexin V–FITC and PI were then added, according to the manufacturer’s instructions, and incubated for 30 min in the dark at 20–25 °C. Acquisition was done on a FACS Calibur flow cytometer (BD) and analysed with CellQuest software. Miltefosine, an established inducer of apoptosis in Leishmania parasites (20 µM, 24 h), served as the positive control (Paris et al., 2004).

Measurement of mitochondrial membrane potential of L. donovani promastigotes. Mitochondrial membrane potential was measured using JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide), a cell-permeable dye that exists in a monomeric form that on entering the cytoplasm emits a green fluorescence. Subsequently, on entering the mitochondria it forms J-aggregates and emits a red fluorescence. The ratio between red and green fluorescence, i.e. 585/530 nm, determines the mitochondrial membrane potential, m (Reers et al., 1995). After being treated with AVL (87.5 µg ml^–1) for 0–24 h, promastigotes were centrifuged (3500 r.p.m. x 10 min). The cells were resuspended in PBS containing JC-1 (10 µg; Molecular Probes) and incubated at 37 °C for 7 min. Analysis for mean green and red fluorescence intensity was done using FACS Calibur and CellQuest software.

Release of cytochrome c from mitochondria of L. donovani promastigotes. Promastigotes were treated with AVL (87.5 µg ml^–1, 0–16 h) and initially loaded with mitotracker deep red as per the manufacturer’s instructions (Molecular Probes). The cells were then fixed and permeabilized with cytofix and cytoperm, respectively. After nonspecific blocking with 2 % PBS–BSA, cells were incubated with murine anti-cytochrome c followed by FITC-labelled anti-murine IgG and viewed under a TCS-SP confocal microscope (Leica). At least 20 microscopic fields were observed for each sample.

Analysis of the nuclear morphology of promastigotes. Oligonucleosomal fragmentation of AVL-treated parasites was identified microscopically using the fluorescent nuclear stain Hoechst 33258. Briefly, promastigotes were incubated with AVL (87.5 µg ml^–1) for 0–72 h, cells were then centrifuged at each time point and the resultant pellet was loaded with Hoechst (5 µg ml^–1) for 30 min at 20–25 °C, attached on poly-L-lysine-coated glass slides, mounted in glycerol containing 1,4-diazabicyclo[2.2.2]octane and examined under a confocal microscope (Leica). At least 20 microscopic fields were observed for each sample.

In situ detection of DNA fragmentation by terminal deoxynucleotidyltransferase (TdT)-mediated dUTP end labelling (TUNEL). DNA fragmentation within the cell was analysed using the Apo-Direct kit (BD) following the manufacturer’s instructions. Briefly, promastigotes were treated with AVL (87.5 µg ml^–1 for 0, 6, 24, 48 and 72 h); cells were then washed twice in PBS and fixed in paraformaldehyde (1 %) for 30 min. Cells were again washed in PBS and kept in chilled ethanol (70 %) for 30 min on ice following which they were washed twice, reacted with TdT enzyme in reaction buffer and stained with PI and dUTP–FITC (Apo-Direct kit). Finally, cells were resuspended in PBS before acquisition on a FACS Calibur and analysed by CellQuest software.

Oligonucleosomal DNA fragmentation assay. Analysis of the presence of DNA fragments generated as a function of cell death was performed using the Apoptotic DNA Ladder kit (Roche) as per the manufacturer’s instructions. Briefly, genomic DNA was isolated from promastigotes exposed to AVL (87.5 µg ml^–1 for 0, 24, 48 and 72 h) or miltefosine (40 µM for 24 h). Extracted DNA was quantified spectrophotometrically by the absorbance ratio of 260/280 nm and DNA (2–3 µg per lane) was run on an agarose (1 %) gel containing ethidium bromide in TBE buffer (50 mM; pH 8.0) for 1.5 h at 75 V and visualized under UV light. Miltefosine-treated promastigotes (40 µM, 24 h) were used as the positive control.

Generation of reactive oxygen species (ROS) in promastigotes. To measure AVL-mediated generation of ROS by promastigotes, H₂DCFDA, a non-fluorescent dye, was used based on the existing knowledge that it is converted by free radicals to a fluorescent dye, dichlorofluorescein (Wan et al., 1993). Briefly, promastigotes were incubated with AVL (87.5 µg ml^–1) at 22 °C for different time points (0–72 h). At each time point, cells were fixed in paraformaldehyde (4 %), nonspecific binding sites were blocked with 2 % PBS–BSA and then probed with H₂DCFDA (10 µM) for 30 min at 20–25 °C. Cells were analysed for intracellular ROS by FACS Calibur using CellQuest software.

Flow cytometric assessment of intracellular Ca²⁺ in L. donovani promastigotes. Changes in intracellular Ca²⁺ were monitored using the fluorescent probe fluo-3/AM as previously described (Mukherjee et al., 2002). Briefly, AVL-treated promastigotes (87.5 µg ml^–1 for 0, 6, 12, 24, 48 and 72 h) were loaded for 30 min at 20–25 °C with fluo-3/AM (5 µM) containing pluronic acid F127 (1 µM) for proper dispersal and sulfinpyrazone (0.25 mM), an organic anion transport inhibitor, to inhibit leakage of the fluo-3 dye. Fluorescence intensity was expressed as the increase in fluorescence with respect to baseline fluorescence intensity before stimulation. For detection of fluo-3 staining in promastigotes, fluo-3-labelled cells were excited with a 488 nm laser and images were collected at 530 nm using a FACS Calibur. Promastigotes treated with Ca²⁺ ionophore or Ca²⁺ ionophore with EGTA served as positive and negative controls, respectively.

Determination of protease activity in AVL-treated promastigotes. To study the role of caspases and other proteases in AVL-induced death, exponential-phase promastigotes were harvested and resuspended in Medium A. Cells were seeded (1x10⁵ in 250 µl per well) in 96-well tissue culture plates and pre-incubated for 2 h with one of the protease inhibitors, namely aprotinin (3 µg ml^–1), leupeptin (100 µM), phenylmethylsulfonylfluoride (PMSF, 1 mM), pepstatin (10 µM), trypsin inhibitor (1 mM), EDTA (10 mM), EGTA (10 mM) or Z-Val-Ala-DL-Asp (methoxy)-fluoromethylketone (Z-VAD-FMK, 100 µM). Subsequently, AVL (200 µg ml^–1) was added and co-incubated for a further 72 h. The viability of promastigotes was evaluated using the colorimetric ‘modified MTT assay’ (Dutta et al., 2005).

Statistical analysis. In vitro antileishmanial activity was expressed as the IC₅₀ by linear regression analysis. Values are mean±SD from at least three independent experiments in duplicate.

	RESULTS AND DISCUSSION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Determination of the IC₅₀ of AVL-mediated death in L. donovani promastigotes

The viability of promastigotes was evaluated by the ‘modified MTT assay’ wherein the conversion of MTT to formazan by mitochondrial enzymes serves as an indicator of cell viability; accordingly, a decrease in formazan production indicates decreased cell viability and vice versa (Dutta et al., 2005). Treatment of promastigotes with AVL demonstrated a dose-dependent inhibition of parasite growth and the IC₅₀ in AG83 (MHOM/IN/83/AG83) promastigotes was 110 µg ml^–1 (Fig. 1).

View larger version (12K): [in this window] [in a new window]   Fig. 1. Analysis of the antileishmanial activity of AVL. Promastigotes (MHOM/IN/83/AG83; 5x104 in 250 µl per well) were incubated with increasing concentrations of AVL (0–300 µg ml–1) for 72 h and the MTT assay was performed as described in Methods. Each point corresponds to the mean±SD of at least three experiments in duplicate.

Accordingly, in order to study whether the mechanism of cell death triggered by AVL is via apoptosis or necrosis, AVL-treated promastigotes (87.5 µg ml^–1 for 0, 6, 16 and 24 h) were double-stained with FITC-conjugated annexin V and PI. As shown in Fig. 2, a significant percentage (51.0 %) of promastigotes of AVL-treated cells (24 h) stained positive for annexin V (Fig. 2, lower and upper right quadrants) compared to only 2.0 % in untreated cells (Fig. 2). This was comparable with apoptosis triggered by miltefosine (64.5 %; Fig. 2, lower and upper right quadrants), which served as a positive control based on previous studies confirming that its antileishmanial activity is mediated via apoptosis (Paris et al., 2004). The percentage of PI-stained (upper left quadrant) or double-stained (upper right quadrant) cells ranged from 2 to 11 % in all experimental sets, indicating that AVL exerts its leishmanicidal activity primarily via apoptosis.

View larger version (27K): [in this window] [in a new window]   Fig. 2. Externalization of phosphatidylserine in AVL-treated promastigotes. Promastigotes (MHOM/IN/83/AG83) were incubated with AVL (87.5 µg ml–1) or miltefosine (20 µM) for 24 h, co-stained with PI and annexin V–FITC and analysed by flow cytometry as described in Methods. This is a representative profile of at least three experiments.

As shown in Fig. 3, a 3 h treatment with AVL (87.5 µg ml^–1) caused hyperpolarization evidenced by a 33.3 % increase in the 585/530 nm ratio of AVL-treated versus untreated cells, being 6.37±0.19 versus 4.78±0.23, respectively. This was sustained up to 6 h, being 6.22±0.15 versus 4.71±0.21. However, at 10 h, the degree of hyperpolarization decreased, the 585/530 nm ratio of treated versus non-treated cells being 3.81±0.27 versus 3.21±0.31, respectively. At 14 h, AVL induced hypopolarization of m as the 585/530 nm ratio of treated versus non-treated cells was 2.89±0.38 versus 3.66±0.31, respectively. At 24 h, a further decrease in the m was observed, the 585/530 nm ratio in treated versus non-treated cells being 2.08±0.23 versus 3.98±0.17, respectively (Fig. 3). Taken together, the data indicate that AVL up to the 10th hour caused mitochondrial membrane hyperpolarization, which was followed by a sustained hypopolarization thereafter (Fig. 3).

View larger version (15K): [in this window] [in a new window]   Fig. 3. Changes in mitochondrial membrane potential following treatment with AVL. Promastigotes (MHOM/IN/83/AG83) were incubated at 22 °C in the absence (filled bars) or presence (87.5 µg ml–1; open bars) of AVL for different time points, washed in PBS and probed with JC-1 (10 µg in 100 µl per well) and analysed by flow cytometry as described in Methods. The 585/530 nm ratio, i.e. J-aggregates, in the mitochondria versus monomers in the cytosol represents the m.

View larger version (83K): [in this window] [in a new window]   Fig. 4. Presence of cytosolic cytochrome c in promastigotes following AVL treatment. Promastigotes (MHOM/IN/83/AG83) were incubated with AVL (87.5 µg ml–1) for different time points and the binding of FITC-labelled cytochrome c was analysed by confocal microscopy as described in Methods. Mitotracker was used to identify the mitochondria.

View larger version (72K): [in this window] [in a new window]   Fig. 5. Analysis of nuclear blebbing of AVL-treated promastigotes. Promastigotes (MHOM/IN/83/AG83) were incubated with AVL (87.5 µg ml–1) for different time points (0, 24, 48 and 72 h), probed with Hoechst 33258, and analysed by confocal microscopy as described in Methods. (a) Fluorescence panel; (b) phase-contrast panel; (c) overlay of the previous two.

This in situ finding was corroborated by oligonucleosomal DNA fragmentation analysis of promastigotes treated with AVL (87.5 µg ml^–1). A characteristic ladder was observed and a time-dependent increase in fragmentation of nuclear DNA was evident as detectable by gel electrophoresis. Promastigotes treated for 72 h with AVL showed a similar degree of fragmentation to that observed in miltefosine-treated cells (40 µM, 24 h).

Late-stage events in the process of apoptosis in Leishmania include nuclear condensation and DNA nicking (Verma & Dey, 2004). As this was evident in AVL-treated promastigotes, it strongly suggested that AVL triggers an apoptosis-like death in Leishmania parasites. Formation of a DNA ladder (200 bp) is the hallmark of apoptosis, and as AVL-treated promastigotes showed a similar profile, this provided corroboratory evidence that AVL promoted apoptosis in Leishmania promastigotes.

AVL failed to generate ROS in promastigotes

To investigate whether AVL caused ROS generation within promastigotes, a fluorescent probe, H₂DCFDA, was used. This probe primarily detects H₂O₂ and hydroxyl radicals, O₂H^–, and fluoresces after forming dichlorofluorescein; therefore, an increase in signal indicates augmented generation of H₂O₂ and hydroxyl radicals (Wan et al., 1993). Although an inherent basal level of ROS production in promastigotes was detectable (MFI=10.87), the addition of AVL (6, 12, 16, 24, 48 and 72 h) failed to induce any changes in the MFI, being 10.91, 11.43, 11.06, 9.11, 11.29 and 9.59, respectively, indicating that AVL did not trigger ROS generation in promastigotes. Although kinetoplastid parasites share features reminiscent of mammalian nucleated cell apoptosis, the pathways (induction/execution) may be different at the molecular level. An established event in most apoptotic cells is generation of ROS in the cytosol, which directs the cell and its neighbouring cells towards the path of death (Chipuk & Green, 2005). However, with AVL treatment, no ROS generation was evident.

Intracellular Ca²⁺ was unaltered by AVL treatment

An elevation in intracellular Ca²⁺ for endonuclease activity is generally considered a requirement for PCD. In promastigotes, calcium ionomycin increased intracellular Ca²⁺ levels from baseline levels as evidenced by the given fluorescence of fluo 3/AM, the MFI being 88.35 versus 21.93, respectively. The addition of EGTA, a Ca²⁺-specific metalloprotease, decreased the MFI to 1.56. However, AVL (87.5 µg ml^–1; 6, 12, 24, 48 and 72 h) failed to change the Ca²⁺ levels in promastigotes, which remained close to baseline levels, the MFI being 21.30, 26.09, 19.76, 16.41 and 17.29, respectively.

Calcium is an important element in the progression towards cell death, as most endonucleases require the presence of Ca²⁺ to cleave DNA strands, and therefore in apoptosis, elevation of Ca²⁺ is consistently increased. However, with regard to AVL, the absence of a measurable alteration in Ca²⁺ despite the presence of DNA cleavage as confirmed by the TUNEL assay and DNA laddering, suggests that either the existing cellular Ca²⁺ is sufficient for endonuclease activity or possibly these endonucleases are Ca²⁺-independent (Kawabata et al., 1996).

Antipromastigote activity of AVL was via a caspase-independent pathway

To evaluate the role of caspases and other proteases in AVL-induced apoptosis, promastigotes were preincubated (2 h) with or without protease inhibitors, namely aprotinin, leupeptin, PMSF, pepstatin, trypsin inhibitor, EDTA and EGTA as well Z-VAD-FMK, followed by the addition of AVL (200 µg ml^–1, 72 h). The viability was measured using the modified MTT assay.

The percentage of AVL-induced promastigote killing was 77.35±6.07 %. This remained unchanged in the presence of serine protease inhibitors (aprotinin, 83.84±5.31 %; PMSF, 76.46±6.23 %), a serine and cysteine protease inhibitor (leupeptin, 77.14±4.28 %), an aspartic protease inhibitor (pepstatin, 74.70±3.89 %), metalloprotease inhibitors (EDTA, 70.74±4.12 %; EGTA, 75.91±3.54 %) or trypsin inhibitor (82.10±2.83 %). Even the broad-spectrum caspase 3 inhibitor Z-VAD-FMK failed to attenuate AVL-induced promastigote killing (80.93±3.59 %), indicating that AVL-induced apoptosis is caspase-independent. It has been reported that caspase-mediated cell death causes formation of apoptotic bodies and shrinkage of the cell surface, which are absent in caspase-independent PCD (Chipuk & Green, 2005).

Studies pertaining to the signalling pathway, downstream of the formation of a cleavable complex, that ultimately leads to an increase in the number of apoptotic cells is still not clearly established. Generally, reduced cytochrome c attaches with apoptosis protease activating factor to activate caspase 3 downstream in metazoan cells (Green & Kroemer, 2004). However, caspase activation is not mandatory as studies with murine embryonic fibroblasts on exposure to UV irradiation showed release of cytochrome c into the cytosol in the absence of caspase activation (Carmen et al., 2006). In Leishmania, the role of caspases in apoptosis remains controversial as stress promoted death is caspase-independent (Zangger et al., 2002) whereas camptothecin causes a caspase-dependent death (Sen et al., 2004). In our experiments, AVL-induced reduction in cell viability of promastigotes remained unchanged in the presence of a broad-spectrum caspase inhibitor (Z-VAD-FMK), implying that AVL-mediated parasite death is via a caspase-independent pathway. Alternative pathways resulting in caspase-independent apoptotic cell death have been reported in promastigotes and amastigotes of L. major and L. mexicana upon serum depletion (Debrabant et al., 2003). Following treatment with AVL, the decrease in cell viability remained unaltered in the presence of a number of cell-permeable protease inhibitors, virtually eliminating the possibility of proteases being involved in the apoptotic pathway. Therefore, our results suggest that AVL caused a protease-independent PCD in L. donovani promastigotes.

Apoptosis-like changes have been reported for mediating the leishmanicidal action of miltefosine (Verma & Dey, 2004), amphotericin B (Lee et al., 2002) and camptothecin (Sen et al., 2004). The efficacy of AVL against promastigotes of L. braziliensis, L. mexicana, L. tropica, L. major and L. infantum has recently been reported (Dutta et al., 2007). In this study, we have shown that AVL has a similar leishmanicidal effect against promastigotes of L. donovani (IC₅₀=110 µg ml^–1; Fig. 1). To clarify the mode of action of AVL against L. donovani, using biochemical and morphological approaches we have demonstrated that AVL-induced cell death shared several phenotypic features observed with metazoan apoptosis (Debrabant et al., 2003), which included phosphatidylserine exposure (Fig. 2), hypopolarization of mitochondrial potential (Fig. 3), release of cytochrome c into the cytosol (Fig. 4), nuclear blebbing by Hoechst staining (Fig. 5), in situ TUNEL staining of nicked DNA and oligonucleosomal DNA fragmentation. Taken together with the combined use of several techniques, including annexin V staining, changes in mitochondrial potential, release of cytochrome c into the cytosol and DNA fragmentation, we have conclusively proven that AVL induced a caspase-independent PCD in promastigotes that notably shares some, but not all, the classical features of apoptosis observed in higher eukaryotes. It is envisaged that study of the major pathways involved in Leishmania apoptosis-like death would help provide insight for future design of newer chemotherapeutic strategies.

	ACKNOWLEDGEMENTS

 
The work received financial assistance from the Council of Scientific and Industrial Research and the Life Sciences Research Board (LSRB), India. A. D. is the recipient of a Senior Research Fellowship from the Indian Council of Medical Research, Government of India, and Dr Suman Bandyopadhyay received a Senior Research Fellowship from Council of Scientific and Industrial Research, India.

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