Experimental Parasitology 123 (2009) 258–264

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Experimental Parasitology

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Leishmania donovani: A glycosyl dihydropyridine analogue induces apoptosislike cell death via targeting pteridine reductase 1 in promastigotes

Jaspreet Kaur a, Biswajit Kumar Singh b, Rama Pati Tripathi b, Prashant Singh c, Neeloo Singh a,*

a Drug Target Discovery & Development Division, Central Drug Research Institute, Chattar Manzil Palace, P.O. Box No. 173, Lucknow 226001, Indiab Medicinal and Process Chemistry Division, Central Drug Research Institute, Chattar Manzil Palace, P.O. Box No. 173, Lucknow 226001, Indiac Department of Chemistry, D.A.V. (P.G.) College, Dehradun 248001, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 1 June 2009Received in revised form 24 July 2009Accepted 27 July 2009Available online 6 August 2009

Keywords:Leishmania donovaniPteridine reductase 1 (EC 1.5.1.33)Antifolate chemotherapyFlow cytometryCell cycle arrest

0014-4894/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.exppara.2009.07.009

* Corresponding author. Fax: +91 522 22623405.E-mail address: neeloo888@yahoo.com (N. Singh).

Targeting of pteridine reductase 1 (PTR1) in Leishmania is essential for development of successful antif-olate chemotherapy. In search for specific inhibitors of PTR1 we have previously reported phenyl 1,4-dihydropyridine ring as the lead structure showing antileishmanial efficacy in vitro and by the oral routein vivo. In this study, we present programmed cell death inducing potential of this glycosyl dihydropyr-idine analogue (2,6-dimethyl-4-(3-O-benzyl-1,2-O-isopropylidene-b-L-threo-pentofuranos-4-yl)-1-phe-nyl-1,4-dihydro-pyridine-3,5-dicarboxylic acid diethyl ester). Flow cytometric analysis revealed thatthis analogue induces cell cycle arrest at G2/M phase with subsequent increase in sub-G1 peak. Incuba-tion of Leishmania promastigotes with this analogue causes exposure of phosphatidylserine to the outerleaflet of plasma membrane, formation of reactive oxygen species, depolarization of mitochondrial mem-brane potential and concomitant nuclear alterations that included DNA fragmentation. The results fromthis study on promastigotes give important lead to investigate further in intracellular amastigotes, thebiologically relevant parasite stage in host macrophages.

� 2009 Elsevier Inc. All rights reserved.

1. Introduction

Leishmaniasis occurs as cutaneous, mucosal and visceral (themost severe) clinical forms and remains a major health problemin the tropics and subtropics, threatening almost 350 million peo-ple in 88 countries (Murray et al., 2005). Approximately 50% of theworld’s cases of visceral leishmaniasis (VL) occur in the Indian sub-continent (Desjeux, 2004). To date, there are no vaccines againstleishmaniasis and therefore treatment relies solely on chemother-apy. The drugs recommended for the treatment of VL (pentavalentantimonials, pentamidine, amphotericin B and its lipid formula-tions, paromomycin, allopurinol and miltefosine) have limitations,including development of resistance, parenteral administration,long courses of treatment, toxic side effects, and high costs (Per-ez-Victoria et al., 2003; Sundar and Chatterjee, 2006). Therefore,there is continued interest in development of effective, safe andcheaper chemotherapeutic agents for treating leishmaniasis.

Enzymes, metabolites or proteins present in the parasite but ab-sent from their mammalian host are considered as ideal targets forrational drug design. Thus pteridine reductase 1 (PTR1, EC 1.5.1.33)of Leishmania has been perceived as an excellent target owing tothe unusual salvage of pterin from the host, while the host can syn-

ll rights reserved.

thesize pterin derivatives de novo from Guanidine Tri Phosphate(GTP) and lack PTR1 activity (Nichol et al., 1985). We have focusedon a virulent clinical isolate of Leishmania donovani when investi-gating the role of pteridine metabolism for antiparasite chemo-therapy (Singh, 2002). In search for specific inhibitors of PTR1(Accession No. AY547305) we have previously reported the phenyl1,4-dihydropyridine ring as a lead structure showing antileishma-nial efficacy in vitro with an IC50 of 90 lM (Kumar et al., 2008) andin vivo with an EC50 of 25 mg/kg body wt (unpublished results).

The principal biological activity associated with 1,4-dihydro-pyridine skeleton is as Ca2+ channel blockers with their role asdrugs for the treatment of cardiovascular diseases and hyperten-sion (Bossert et al., 1981; Nakayama and Kasoka, 1996). Thisskeleton is frequently seen in many vasodilator, bronchiodilator,anti-atherosclerotic, antitumor, hepatoprotective and antidiabeticagents (Saushins and Duburs, 1988; Mager et al., 1992). They arealso known as neuroprotectants, anti-platelet treatment of aggre-gators and are important in Alzheimer’s disease as antiischemicagents (Klusa, 1995; Bretzel et al., 1993; Boer and Gekeler, 1995;Priego et al., 1992). Interest in 1,4-dihydropyridine chemistry andbiochemistry also relates to nicotinamide dinucleotide (NADH), aco-enzyme, and its unique ability to reduce many functionalgroups in biological systems. 1,4-Dihydropyridine derivatives havebeen reported to be active against experimental VL and triggeredprogrammed cell death (PCD) of the parasites (Palit and Ali, 2008).

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PCD mechanisms are known to be operative in kinetoplastidparasites of the genera Trypanosoma (Szallies et al., 2002) andLeishmania in response to various chemotherapeutic stimuli suchas amphotericin B (Lee et al., 2002), staurosporine (Arnoult et al.,2002), camptothecin (Sen et al., 2004), miltefosine (Paris et al.,2004), aloe vera leaf extracts (Dutta et al., 2007a), artemisinin(Sen et al., 2007), racemoside A (Dutta et al., 2007b), and curcumin(Das et al., 2008).

In this study, our objective was to evaluate the cell deathmechanism of glycosylated dihydropyridine analogue (GDA) (2,6-dimethyl-4-(3-O-benzyl-1,2-O-isopropylidene-b-L-threo-pentofur-anos-4-yl)-1-phenyl-1,4-dihydro-pyridine-3,5-dicarboxylic aciddiethyl ester) against L. donovani clinical isolate. We report that,in promastigotes of L. donovani, cytotoxicity of GDA leads to PCD,which were associated with cellular-cycle alterations in G1, Sand G2/M phases and changes in the mitochondrial membrane po-tential culminating in DNA fragmentation.

2. Materials and methods

2.1. Materials

M-199 medium and fetal bovine serum (FBS) were obtainedfrom Gibco-BRL, dimethylsulphoxide (DMSO) from SRL, ethanolfrom Merck, ApoAlert annexin V-FITC apoptosis kit from Sigma,Apoptotic DNA ladder kit, 20,70-dichlorofluorescein diacetate(H2DCFDA), propidium iodide (PI) and MitoTracker deep red fromMolecular Probes. All other chemicals were from Sigma unlessstated.

2.2. Candidate inhibitor

GDA (2,6-dimethyl-4-(3-O-benzyl-1,2-O-isopropylidene-b-L-threo-pentofuranos-4-yl)-1-phenyl-1,4-dihydro-pyridine-3,5-dicarbox-ylic acid diethyl ester) was prepared following an earlier protocol(Tewari et al., 2004) and characterized on the basis of its spectro-scopic data and microanalysis (Kumar et al., 2008).

2.3. Parasite culture

Isolates R-5 and 2001 used in the present study were obtainedfrom patients with VL. Based on their clinical response to sodiumantimony gluconate (SAG), they were classified as SAG sensitive(2001) or SAG resistant (R-5) (Singh et al., 2003; Dube et al.,2005). They were routinely cultured at 25 �C in M-199 mediumsupplemented with 10% heat inactivated fetal bovine serum(HIFBS), 100 U penicillin and 100 lg streptomycin/ml.

2.4. Analysis of externalized phosphatidylserine in L. donovanipromastigotes by flow cytometry

2 � 106 exponential-phase L. donovani promastigotes wereincubated with GDA (90 lM for 0–96 h). Cells were centrifuged(2000g for 5 min), washed twice in PBS and resuspended in annex-in V binding buffer [10 mM Hepes/NaOH (pH 7.4), 140 mM NaCl,2.5 mM CaCl2]. Annexin V-FITC and PI were then added accordingto the manufacturer’s instructions, and incubated for 30 min inthe dark at 20–25 �C. Data acquisition was carried out on a FACSA-ria flow cytometer (BD Bioscience) and analysed using CellQuestPro software. If there is an alteration of the membrane integrity(due to externalization of phosphatidylserine), annexin V detectsboth early- and late-apoptotic cells. Therefore, the simultaneousaddition of PI, which does not enter healthy cells with an intactplasma membrane, discriminates between early-apoptotic (annex-in V-positive and PI-negative), late-apoptotic (both annexin V and

PI-positive), necrotic (PI-positive) and live (both annexin V and PI-negative) cells.

2.5. Measurement of reactive oxygen species (ROS) level

Intracellular ROS level was measured in treated and untreated L.donovani promastigotes as described previously (Mukherjee et al.,2002). Briefly, log phase promastigotes (2 � 106) after differenttreatments were washed and resuspended in 500 ll of medium M-199 and were loaded with the cell permeant probe 2,7-dichlorodihy-drofluorescein diacetate (H2DCFDA) (10 lM) for 30 min at 20–25 �C,and fluorescence was monitored. The fluorescent probe H2DCFDA isone of the most widely used techniques for direct measuring of theredox state of a cell. It is a cell permeable, relatively non-fluorescentmolecule. It is also extremely sensitive to the changes in the redoxstate of a cell and can be used to follow the changes of ROS over time.Activity of cellular esterases cleaves H2DCFDA into 2,7-dichlorodi-hydrofluorescein (DCFH2). Peroxidases, cytochrome c and Fe2+ canall oxidize DCFH2 to 2,7-dichlorofluorescein (DCF) in the presenceof hydrogen peroxide. Accumulation of DCF in the cells may be mea-sured by an increase in fluorescence at 530 nm when the sample isexcited at 485 nm. Fluorescence at 530 nm can be measured usinga FACSAria flow cytometer and analysed using Cell Quest Pro soft-ware. It is assumed to be proportional to the concentration of hydro-gen peroxide in the cells.

2.6. Measurement of mitochondrial membrane potential

Mitochondrial damage upon treatment with GDA in L. donovanipromastigotes was assessed by flow cytometry using a cell perme-able dye, MitoTracker deep red. MitoTrackers are aldehyde fixablecationic lipophilic fluorochrome that passively diffuse through theplasma membrane of viable cells and are selectively sequestered inmitochondria with an active membrane potential and permit theexamination of the membrane potential in adherent cells (Hau-gland et al., 1996). Promastigotes were treated with GDA (90 lMfor 0–96 h), washed in PBS and loaded in dark for 30 min withMitoTracker deep red (10 lM) according to the manufacturer’sinstructions. Analysis for mean fluorescence intensity (MFI) wasdone using FACSAria and analysed using CellQuest Pro software.

2.7. Oligonucleosomal DNA fragmentation assay

To analyse the presence of DNA fragments generated as a func-tion of cell death, total cellular DNA from promastigotes exposed toGDA (90 lM for 0–96 h) were isolated according to manufacturer’sinstructions (Apoptotic DNA ladder kit from Molecular Probes). Ex-tracted DNA was quantified spectrophotometrically by the absor-bance ratio of 260/280 nm and DNA (5 lg/lane) was separated byelectrophoresis on 1% agarose gel containing ethidium bromidein TBE buffer (50 mM; pH 8.0) for 1.5 h at 75 V, visualized underUV light and photographed using a gel documentation system(Alpha Imager).

2.8. DNA content analysis by flow cytometry

2 � 106 log phase L. donovani promastigotes were treated withGDA (90 lM for 0–96 h) were harvested by centrifugation at2000g, for 5 min at 4 �C. Cells were washed once in 1 ml PBS andthen fixed by incubation in 70% ethanol: 30% PBS for 1 h at 4 �C.Prior to analysis, fixed cells were harvested by centrifugation at1000g, for 10 min at 4 �C, washed in 1 ml PBS and then resus-pended in 1 ml PBS with RNAse A (100 lg/ml) and propidiumiodide at 10 mg/ml. The cells were incubated at 25 �C for 30 minand then analysed by using a FACSAria flow cytometer. Ten thou-sand cells were analysed for each sample. Cell cycle distribution

260 J. Kaur et al. / Experimental Parasitology 123 (2009) 258–264

was modeled using the ModFit LT software package (Verity Soft-ware House) in accordance with the standards detailed in Hedleyet al. (1993).

2.9. Statistical analysis

Data are presented as mean ± SD. The statistical significance ofdifferences in percentage between treated and untreated was ana-lysed by one-way ANOVA using GraphPad Prism software.

3. Results and discussion

3.1. GDA-treated L. donovani promastigotes show both annexin V andPI binding

Translocation of phosphatidylserine from the inner side to theouter layer of the plasma membrane is a common alteration duringPCD (Mehta and Shaha, 2004). Annexin V, a Ca2+-dependentphospholipid-binding protein with a special affinity for phosphati-dylserine, is a general reagent used to detect the externalization ofphosphatidylserine, thus labeling cells that have lost their mem-brane integrity. Accordingly, to determine whether GDA triggeredcell death followed a similar course, promastigotes treated withGDA (90 lM for 0–96 h) were double-stained with annexin V-FITCand PI. The percentage of GDA-treated promastigotes that were po-sitive only for annexin V gradually decreased, to 68.39%, 65.53%,60.93% and 55.21% at 24, 48, 72 and 96 h, respectively (Fig. 1).However, the number of cells that were both annexin V and PI-po-sitive (upper-right quadrant) gradually increased, from 4.59%,11.58%, 18.00% and 23.77% at 24, 48, 72 and 96 h, respectively(Fig. 1). These observations suggested that GDA induced cell deathby loss of membrane integrity, as shown by the increased PI incor-poration and annexin V binding, indicating advanced apoptoticphase. In contrast, only 0.37% of untreated cells, which served ascontrol, also incubated for 96 h, were annexin V and PI-positive

Fig. 1. Externalization of phosphatidylserine in GDA-treated promastigotes. L. donovancytometry. Undamaged cells were unstained with annexin V-FITC/PI (bottom left quadrpositive with annexin V-FITC and negative with PI (bottom right quadrant). Following thannexin V-FITC and by PI (upper-right quadrant). During advanced apoptosis stages, thetwo other independent experiments produced similar results.

and showed no apoptosis like cell death. The number of cells posi-tive for PI only was negligible at all time points. This indicated thatGDA is a membrane-attacking molecule for L. donovani promastig-otes, acting directly on the plasma membrane, damaging its integ-rity and thus driving PCD signals.

3.2. GDA induces formation of ROS in L. donovani promastigotes

To investigate whether GDA caused ROS generation within L.donovani promastigotes, a fluorescent probe, H2DCFDA, was used.This probe primarily detects H2O2 and hydroxyl radicals, O2H�,and fluoresces after forming DCF; therefore, an increase in signalindicates augmented generation of H2O2 and hydroxyl radicals(Wan et al., 1993). Although an inherent basal level of ROS produc-tion in untreated promastigotes was detectable with mean fluores-cence intensity of 11.87, treatment of promastigotes with GDA fordifferent time periods reveals an elevation in ROS generation(Fig. 2). The level of hydroxyl radicals in GDA treated cells increasesfrom 11.94, 14.72, 22.03, 35.22 and 37.00 at 18, 24, 48, 72 and 96 h,respectively, indicating that GDA trigger ROS generation in prom-astigotes. An established event in most apoptotic cells is genera-tion of ROS in the cytosol, which directs the cell and itsneighbouring cells towards the path of death (Chipuk and Green,2005).

3.3. GDA depolarizes the mitochondrial membrane potential ofL. donovani promastigotes

The loss of mitochondrial membrane potential is a characteristicfeature of metazoan apoptosis and has been observed to play a keyrole in drug-induced death in protozoans such as Leishmania (Senet al., 2004). L. donovani promastigotes were treated with GDA,and following treatment, membrane potential was determined bycytofluorometric measurement of the mitochondrial dependent up-take and retention of MitoTracker Red (CMXRos) into mitochondria.

i promastigotes were incubated with 90 lM GDA for 0–96 h and analysed by flowant). After incubation for 24 h, a significant number of apoptotic cells were stainede increasing of time duration 48–96 h, advanced apoptotic cells stained positive bycells were no longer viable. Data are presented as means ± SD from triplicates and

Fig. 2. Measurement of GDA induced generation of ROS. Generation of ROS was measured using the fluorescent dye 2,7-dichlorodihydrofluorescein diacetate after treatmentwith 90 lM GDA for 0–96 h and its fluorescence was measured using a flow cytometer. Data are presented as means ± SD from triplicates and two other independentexperiments produced similar results.

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Treatment with 90 lM GDA resulted in reduction of MitoTrackerpositive cells from 94.2% (control) to 77.1%, 76.8%, 74.9%, 35.3%and 24.9% at 18, 24, 48, 72 and 96 h, respectively (Fig. 3). Taken to-gether, these results indicated that exposure to GDA caused sus-tained mitochondrial membrane depolarization for up to 96 h,which may be due to imperfect mitochondrial function.

Fig. 3. Changes in mitochondrial membrane potential following treatment with GDA. L.for 30 min with MitoTracker (10 lM) and analysed by flow cytometry. Data are presentesimilar results.

3.4. GDA induces genomic DNA fragmentation in L. donovanipromastigotes

During apoptosis the cleavage patterns of genomic DNA are typ-ical of internucleosomal DNA digestion by an endogenous nucleasethat is considered as a hallmark of late events in the overall apop-

donovani promastigotes were incubated with 90 lM GDA for 0–96 h, loaded in darkd as means ± SD from triplicates and two other independent experiments produced

Fig. 4. Analysis of DNA fragmentation in GDA-treated L. donovani promastigotes.gDNA (10 lg) isolated from L. donovani promastigotes which have been treatedwith GDA (90 lM for 24, 48, 72 and 96 h) were resolved on 1% agarose gel. Crepresents DNA from untreated cells. (Apoptotic DNA ladder kit from MolecularProbes.)

262 J. Kaur et al. / Experimental Parasitology 123 (2009) 258–264

tosis process (Compton, 1992). Treatment of promastigotes withGDA for 24, 48, 72 and 96 h revealed a DNA ladder like pattern. Nu-clear DNA fragmentation into oligonucleosomal-sized fragments(720, 360 and 180 bp), a typical feature of apoptotic cells, wasreadily visible in 72 h (Fig. 4).

Fig. 5. GDA induced increase in sub-G1 peak of L. donovani promastigotes. DNA conteanalysed by flow cytometry. Data are presented as means ± SD from triplicates two oth

3.5. GDA-mediated cell-cycle alteration on L. donovani promastigotes

The least expensive and most rapid discrimination of apoptoticcells is based on DNA content analysis. This is an established ap-proach for screening drug effects in vitro (Darzynkiewicz et al.,2000). In addition to the enumeration of apoptotic cells offeredby this method, the cell cycle specific effects can easily be recog-nized from DNA content histograms of the non-apoptotic cell pop-ulations. We performed a cell-cycle analysis by flow cytometryafter PI staining of the parasites incubated for 0–96 h at 90 lM ofthe GDA. As can be seen from Fig. 5, the GDA induced a decrease(24 h) in promastigotes in both G1 and S phases relative to the con-trol-untreated samples. GDA reduced the percentages of L. dono-vani promastigotes in G1 and S phases by a factor ofapproximately 2 (G1: 78.53% vs. 39.93%; S: 7.23% vs. 3.74% for con-trol- and GDA-treated promastigotes, respectively). In addition, theGDA increased the percentage of promastigotes in G2/M phase by afactor of 4.5 relative to untreated promastigotes (14.28% vs.57.25%, respectively). The time dependent effect on G2/M arrestin promastigotes was largely at the expense of G0/G1-phase cellswith a non-significant change in S-phase cell population comparedwith the untreated promastigotes. The drug induced cell cycle per-turbations such as decrease in the G1 phase have been reported tocorrelate with a response to chemotherapy (Briffod et al., 1992).G2/M is a crucial period for further cellular proliferation. Drug in-duced G2/M arrest is associated with DNA double strand breaksand extensive chromosome damage (Sorenson and Eastman,1988).

To examine whether the GDA induced inhibition of cell prolifer-ation and induction of G2/M arrest in promastigotes leads to theinduction of apoptosis we next incubated the cells with the sameconcentration for 48–96 h. GDA treatment resulted in 26.51%,53.73% and 31.93% sub-G1 peak, characteristic of apoptosis, at48, 72 and 96 h, respectively (Fig. 5). This suggests that a checkin the cell cycle could initiate late events of PCD such as nuclearcondensation and DNA nicking. However, at 18 h, the proportion

nt was analyzed after treatment with 90 lM GDA for 0–96 h, stained with PI ander independent experiments produced similar results.

Fig. 6. Proposed mechanism for the GDA induced apoptosis in L. donovanipromastigotes.

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of cells in different phases of the cell cycle was comparable in bothsets. This finding indicated that nuclear changes are switched onby alterations in the cell membrane and mitochondria and there-fore can be considered as a relatively late phase of PCD.

This study establishes that the leishmanicidal activity of GDA,targeted against PTR1, triggers promastigotes to undergo PCDand activate a cascade of molecular events leading to apoptosis(Fig. 6), which culminates in a cessation of biological activity ofthe targeted enzyme which was not a result of necrosis inducedby an overdose of GDA as a cytotoxic agent.

GDA induced changes in mitochondrial membrane permeabilitywhich, in turn, leads to release of cytochrome c into the cytosol andconsequent activation of caspases, the principal effector moleculesof apoptosis (Debrabant et al., 2003). As a result, it activates DNase,which causes nuclear DNA fragmentation resulting apoptosis like

cell death. Nuclear DNA degradation by endonuclease G upon re-lease from mitochondria (Gannavaram et al., 2008) might be in-volved in PCD.

GDA failed when tested against established resistant clinicalisolates collected from the disease endemic area (Singh et al.,2003; Kothari et al., 2007). Differential sensitivity of a drug resultsfrom the differences in net drug accumulation regulated by influxand/or efflux pathways. The model for antimonial drug resistance(Ouellette et al., 2001; Haimeur et al., 2000) depicted in Fig. 6,can be applied since a clinical resistant isolate has elevated levelof thiols (Mandal et al., 2007; Sarkar et al., 2009), there is activeextrusion of the GDA thiol adduct by MRP pump, lowering theintracellular concentration of the GDA to sub-toxic levels.

Although the current study has been conducted in promastig-otes, the insights obtained into the mechanism of cell death pro-duced by GDA will be valuable in delineating its leishmanicidalactivity in intracellular amastigotes.

Acknowledgments

We are thankful to Mr. A.L. Vishwakarma, Technical Officer SAIFdivision for his help regarding the flow cytometry work. We aregrateful to Department of Biotechnology, India (Grant No. BT/PR5452/BRB/10/430/2004) for financial assistance for this study.

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