1
Induction of mitochondrial dysfunction and oxidative stress in Leishmania donovani by 1
orally active clerodane diterpene 2
3
Manoj Kathuriaa, Arindam Bhattacharjeea, Koneni V. Sashidharab,1, Suriya Pratap Singhb, and 4
Kalyan Mitraa,1* 5
…………. 6
aElectron Microscopy Unit, Sophisticated Analytical Equipment Facility; bMedicinal & Process 7
Chemistry Division, 1Academy of Scientific and Innovative Research, CSIR-Central Drug 8
Research Institute, Lucknow, India. 9
10 11 12 13
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Running Head: Apoptosis induced by clerodane diterpene in Leishmania 16
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* Address correspondence to Kalyan Mitra, [email protected]. 18
M.K. and A.B. contributed equally to this work. 19
This is Communication No. 149/2013/KM from CSIR- Central Drug Research Institute 20
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AAC Accepts, published online ahead of print on 11 August 2014Antimicrob. Agents Chemother. doi:10.1128/AAC.02459-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Abstract 27
This study was performed to investigate the mechanistic aspects of cell death induced by a 28
clerodane diterpene (K-09) in Leishmania donovani promastigotes previously demonstrated to be 29
safe and orally active against Visceral Leishmaniasis (VL). K-09 caused depolarization of the 30
mitochondrion and generation of reactive oxygen species triggering an apoptotic response in L. 31
donovani promastigotes. Mitochondrial dysfunction subsequently resulted in release of 32
cytochrome c into the cytosol impairing ATP production. Oxidative stress caused depletion of 33
reduced glutathione while pre-treatment with anti-oxidant N-acetyl-cysteine (NAC) was able to 34
abrogate oxidative stress. However, NAC failed to restore mitochondrial membrane potential or 35
intracellular calcium homeostasis after K-09 treatment suggesting that generation of oxidative 36
stress is a downstream event relative to the other events. Caspase-3/7-like protease activity and 37
genomic DNA fragmentation were observed. Electron microscopic studies revealed gross 38
morphological alterations typical of apoptosis, including severe mitochondrial damage, pyknosis 39
of nucleus, structural disruption of the mitochondrion-kinetoplast complex, flagellar pocket 40
alterations and displacement of organelles. Moreover, increased number of lipid droplets was 41
detected after K-09 treatment which is suggestive of altered lipid metabolism. Our results indicate 42
that K-09 induces mitochondrial dysfunction and oxidative stress mediated apoptotic cell death in 43
L. donovani promastigotes sharing many features with metazoan apoptosis. These mechanistic 44
insights provide a basis for further investigation towards development of K-09 as a potential drug 45
candidate for VL. 46
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Introduction 51
Leishmaniasis is a neglected tropical vector-borne disease caused by obligate intracellular 52
protozoan parasites of the genus Leishmania. Infection with different species of this genus 53
transmitted through sandfly vectors results in different manifestations of the disease out of which 54
Visceral Leishmaniasis (VL) or ‘kala-azar’, caused by Leishmania donovani is the most severe 55
form and is often fatal. Leishmania spp. have digenetic life cycles involving a flagellated 56
promastigote stage residing in the gut of the sandfly (Phlebotomus sp.) and a non-motile 57
intracellular amastigote stage found in mononuclear phagocytes in the bloodstream of infected 58
individuals (1). Leishmaniasis affects populations from tropical to Mediterranean regions 59
inflicting a heavy burden of morbidity and mortality; according to World Health Organization 60
estimates, 2 million new cases of leishmaniases occur annually with 500,000 cases of VL alone 61
(2). 62
In the absence of any protective vaccination, chemotherapy still remains the mainstay for 63
treatment of leishmaniasis along with effective management against secondary infections. Drugs 64
used in the treatment regimen of VL include pentavalent antimonials, liposomal Amphotericin B, 65
paromomycin, and more recently, the only orally administered drug miltefosine (3). These 66
treatments face severe limitations due to their non-specificity, toxicity, route of administration, 67
cost effectiveness and the tendency to develop resistance (4). Therefore, there is an urgent need 68
for development of new, cheap and easy-to-administer drugs with better safety profiles. The drug 69
discovery effort has now shifted heavily towards natural products due to their limitless variety of 70
novel skeletons for combinatorial modification and their low toxicity. It is interesting to note that 71
~75% of drugs developed against infectious diseases have their origin in nature (5). 72
Apoptosis, a key mechanism for inducing programmed cell death (PCD) has been demonstrated 73
in kinetoplastid protozoans and is no longer considered to be limited to multi-cellular organisms. 74
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Apoptosis is a controlled self-destructing, energy-dependent process exhibiting specific 75
morphological and biochemical features such as cell shrinkage, plasma membrane blebbing, loss 76
of mitochondrial membrane potential, chromatin condensation and nuclear fragmentation (5). 77
Increasing experimental evidence shows that apoptosis-like programmed cell death pathways are 78
functional in Trypanosomatids (7). Apoptosis may be induced by various physiologic (such as 79
nutrient deprivation, heat shock etc.) and chemical (H2O2, chemotherapeutic agents like 80
camptothecin, miltefosine, etc.) stimuli (8-12). Although Leishmania share many biochemical 81
markers with metazoan apoptosis, the molecular machinery involved differs considerably and is 82
not well understood. A better understanding of mechanistic machinery of apoptosis-like PCD in 83
protozoan protists thus would prove immensely beneficial in designing rational chemotherapeutic 84
interventions in a target-dependent manner. 85
In our ongoing efforts to identify and understand the mode of action of new and effective 86
leishmanicidal agents, several natural products are currently being evaluated in our laboratory. 87
Here, we report on the mechanistic aspects of a clerodane diterpene induced cell death in 88
Leishmania donovani. In the present study, we investigated the physiological and ultra-structural 89
alterations in L. donovani promastigotes following administration of a clerodane diterpenoid 90
henceforth designated as K-09 [16α-Hydroxycleroda-3,13(14)Z-dien-15,16-olide] isolated 91
previously from the leaves of Polyalthia longifolia. K-09 was previously reported to be an orally 92
active anti-leishmanial agent working as a DNA topoisomerase inhibitor (13). Our studies reveal 93
that it is capable of inducing promastigote cell death by mitochondrial dysfunction, reactive 94
oxygen species (ROS) generation, elevation of cytosolic Ca2+ and DNA fragmentation. Other 95
apoptotic features such as externalization of phosphatidylserine and caspase-like protease activity 96
were also observed along with increase in the number of cytoplasmic lipid droplets. 97
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Materials and methods 100
2.1 Drugs and chemicals 101
Silica gel column chromatography purified compound (16α-Hydroxycleroda-3, 13 (14) Z-dien-102
15,16-olide), designated as K-09 from hexane extracts of P. longifolia leaves was obtained as 103
reported earlier (12). Stock solution of 5 mg/mL (15.7 mM) was prepared in DMSO and stored at 104
-20°C. N-acetyl cysteine, miltefosine, ionomycin, oligomycin A and Triton X-100 were 105
purchased from Calbiochem, Darmstadt, Germany. JC-1, Carboxy-H2DCFDA, Fluo-4AM, 106
Probenecid, MitoTracker® Deep Red, MitoSOX™ Red and Nile Red were from Molecular 107
Probes, Eugene, OR, USA. All other chemicals were from Sigma-Aldrich, MO, USA unless 108
otherwise stated. 109
110
2.2 Cell culture and treatments 111
L. donovani (strain MHOM/IN/80/DD8) promastigotes were cultured as described previously in 112
Dulbecco’s Modified Eagles Medium (DMEM) supplemented with 10% FBS and gentamicin (40 113
µg/mL) at 26°C (14). After the cell density had reached ~106 cells/mL, the parasites were 114
prepared for drug treatment in fresh medium. K-09 was administered at concentrations of 8 115
μg/mL (IC50: 25 µM) and 16 μg/mL (2×IC50: 50 µM) and incubated for 24h at 26°C. Vehicle 116
control (VC) cells were incubated at the same DMSO concentration comparable to K-09 117
treatments (0.001% v/v). J774A.1 murine macrophages were cultured and infected with L. 118
donovani promastigotes as described earlier (13). 119
120
2.3 Ultra-structural analysis by transmission electron microscopy 121
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Cells were fixed with 4% paraformaldehyde (PFA) and 2% glutaraldehyde in 0.1 M phosphate 122
buffer, pH 7.4 for 4h at room temperature (RT). Samples were then washed in 0.1 M phosphate 123
buffer, post-fixed in 2% OsO4 and encapsulated in agarose. This was followed by dehydration in 124
ascending grades of ethanol, infiltration and embedding in Epon 812 and Araldite plastic mixture 125
and polymerization at 60°C for 24h. Ultrathin sections (50-70 nm) were obtained using an 126
ultramicrotome (Leica Ultracut UCT, Leica Microsystems GmbH, Wetzlar, Germany) and picked 127
up onto 200 mesh copper grids. The sections were double stained with uranyl acetate and lead 128
citrate and observed under a FEI Tecnai-12 Twin Transmission Electron Microscope equipped 129
with a SIS MegaView II CCD camera at 80kV (FEI Company, Hillsboro, OR, USA). At least 400 130
cells were analyzed from the experiments. 131
132
2.4 Analysis of topological alterations by scanning electron microscopy 133
Cells were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer. After washing in phosphate 134
buffer, suspensions were placed on poly-L-lysine coated glass chips and allowed to adhere for 10 135
min at room temperature (RT). Samples were post-fixed in 1% OsO4 and subsequently 136
dehydrated through an ascending ethanol series, critical point dried and coated with Au-Pd 137
(80:20) using a Polaron E5000 sputter coater. Samples were examined in a FEI Quanta 250 SEM 138
at an accelerating voltage of 10 kV using SE detector. Micrographs were taken at magnifications 139
of 5000× and 10000×. About 200 cells from two stubs for each sample were analyzed. Flagellar 140
length and parasite body length of at least 100 cells were measured with xT Microscope Control 141
software (FEI). 142
143
2.5 Confocal microscopy and image analysis 144
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Slides were analyzed under a Carl Zeiss LSM 510 META (Carl Zeiss, Jena, Germany) confocal 145
laser scanning microscope equipped with 405nm diode, Argon multiline (458, 477, 488, 514nm), 146
561nm DPSS and HeNe 633nm lasers. A Plan Apochromat 63×/1.4 N.A. Oil DIC objective lens 147
was used with appropriate excitation and emission filters for imaging. Quantitative analysis of 148
images was performed using Zeiss AIM v4.2. 149
150
2.6 Fluorometric studies 151
Prior to taking fluorometric measurements, cells (107/mL) were transferred in 96 well flat-bottom 152
fluorescence measurement microplates. All experimental data unless stated otherwise were 153
collected with a TECAN Infinite M1000 pro (TECAN group Ltd., Mannedorf, Switzerland) 154
monochromator-based fluorescence microplate reader with top reading, bandwidth = 5.0nm and 155
gain=optimal. 156
157
2.7 Measurement of changes in mitochondrial membrane potential 158
K-09 induced changes in mitochondrial membrane potential (ΔΨm) were measured by the 159
mitochondrial membrane permeable cationic potentiometric vital dye JC-1, which deposits in a 160
ratiometric manner as J-aggregates in the mitochondria of cells with higher ΔΨm, giving red 161
(590nm) fluorescence (indicating healthy cell) and remains as green monomer in the cytoplasm of 162
cells with depolarized mitochondria (i.e. depleted ΔΨm, indicating apoptotic cells). Briefly after 163
harvesting, JC-1 (2 µM), was added to VC and treated cells that were then incubated in darkness 164
for 20 min on poly-L-lysine coated coverslips, mounted and imaged immediately by confocal 165
microscopy. In a subset of VC cells, oligomycin-A (oli-A) (an inhibitor of the F0-F1 ATPase 166
complex) or 50 µM CCCP (a respiratory uncoupler), was added 1h prior to addition of JC-1. The 167
excitation line was 488nm and emission filters were BP 505-550 (green) and LP 575 (red). Mean 168
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fluorescence intensity in each emission channel was measured for ≥20 cells for each case and the 169
530/590 nm ratio was calculated. 170
A subset of the cells following drug treatment were subjected to flow cytometry in a BD FACS 171
Calibur Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) with 488nm excitation 172
and emission filters of 525 ± 15nm and 570 ± 25nm for green and red channels, respectively. Data 173
were analyzed with Cell Quest Pro software with taking 10,000 events as standard. 174
Depolarization event of the mitochondrion was tracked in real time by taking kinetic fluorescence 175
measurements. Cells, pre-stained with 2 µM JC-1 were treated or not with K-09 and CCCP 176
against VC and immediately tracked in the fluorometer (530nm) for 1½h. To test the effect of 177
ROS quenching, N-acetyl-cysteine (NAC) pre-treatment (20 mM, 2h) was given before K-09 178
treatment and kinetic measurements were performed. 179
180
2.8 Estimation of mitochondrial superoxide levels 181
Mitochondrial superoxide levels were estimated with MitoSOX™ Red, a fluorogenic dye specific 182
for mitochondrial superoxide which is cleaved after reacting with O2- produced by the 183
mitochondria and emits red fluorescence upon binding with DNA. The experiment was performed 184
according to manufacturer’s instructions. Confocal microscopy of the cells treated with IC50 dose 185
K-09, 2×IC50 dose K-09 and VC cells was performed by adding 5 μM MitoSOX™ Red solution 186
in PBS to cells adhered on poly-L-lysine coated coverslips and allowed to incubate for 15 min at 187
RT, followed by confocal imaging. Flow cytometry analysis of the above set of cells (107/mL) 188
was also performed following the above protocol keeping the cells in suspension prior to analysis. 189
190
2.9 Monitoring of changes in intracellular Ca2+ levels 191
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Changes in intracellular calcium levels were measured by the fluorometric dye fluo-4 192
acetoxymethyl ester (Fluo 4-AM). Fluo-4AM is a cell-permeant ester that shows large 193
fluorescence yields upon binding intracellular Ca2+. The experiment was performed as described 194
previously (15), with the following modifications. Cells were treated with K-09 (IC50), pretreated 195
with EGTA (2 mM, 2h) before K-09 and with the calcium ionophore ionomycin (5 µM). Next, 196
cells were incubated (45 min, RT) in a cocktail of 5 μM fluo-4AM, 1 μM Pluronic-127 for 197
permeabilization of the dye and 2 mM Probenecid, which prevents of Ca2+ leakage from cells. 198
Post-incubation, cells were washed twice in PBS to remove non-hydrolyzed dye, adhered on poly-199
L-lysine coated coverslips before visualization with confocal microscopy. To observe changes in 200
cytosolic Ca2+ levels in real time cells were pre-stained with Fluo 4-AM as described and then 201
treated with K-09 (IC50), K-09(2×IC50) in the presence of EGTA in the medium (2 mM) and with 202
ionomycin immediately prior to taking kinetic fluorescence measurements. In another set, NAC 203
pre-treatment was given before K-09 treatment and was processed as mentioned. 204
205
2.10 Measurement of ROS levels 206
The fluorogenic marker 5-(and-6)-carboxy-2’,7’-dichlorodihydrofluorescein diacetate (carboxy-207
H2DCFDA) was used for this study, which is a live cell-permeable acetate ester, and upon entry it 208
is cleaved by cellular esterases and reacts with cellular ROS (generated heavily during periods of 209
oxidative stress) to emit fluorescence in the green region by converting into actively fluorescing 210
dichlorofluorescein (DCF). Cellular ROS is quenched by NAC, an amino acid with potent 211
antioxidant capabilities. Cells were treated with K-09 (IC50) and K-09 (IC50) in NAC-pretreated 212
cells (20 mM, 2h) and with 5 mM tert-butyl hydroperoxide (TBHP), a known ROS inducing 213
agent. Cells were then washed once in PBS and adhered to poly-L-lysine coated coverslips 214
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followed by incubation with carboxy-H2DCFDA (10µM, 30m) solution in darkness at RT before 215
processing for confocal imaging. 216
Kinetic fluorometric studies were similarly performed to assess the rate of ROS generation 217
following treatment and the effect of NAC on ROS quenching. Cells were pre-stained with 218
carboxy-H2DCFDA and then treated with K-09 (IC50) with or without NAC-pretreatment and 219
TBHP immediately before taking fluorescence measurements. 220
221
2.11 Measurement of reduced glutathione levels 222
During PCD, elevated generation of free radical is buffered by the intracellular thiol buffer 223
system, the main component of which is the tripeptide glutathione. It exists as reduced (GSH) and 224
oxidized (GSSG) forms, the levels of GSH being drastically reduced in presence of oxidative 225
stress. Reduced glutathione is stained by bimane dyes such as monochlorobimane (16). Cellular 226
Glutathione levels were measured with ApoAlert Glutathione Assay kit (Clontech, Mountain 227
View, CA, USA) according to manufacturer’s instructions with minor modifications. Briefly, 228
cells were harvested and lysed with provided lysis buffer, centrifuged and the supernatant was 229
collected; which was then incubated with 2 mM monochlorobimane (MCB) at 37°C for 3h in 230
darkness. Sample fluorescence was subsequently analyzed on a BMG FLUOstar Omega 231
microplate reader (BMG Labtech, Ortenberg, Germany). 232
233
2.12 Determination of mitochondrial morphology by MitoTracker® Deep Red staining 234
Physical disruption of the parasite mitochondria was investigated with MitoTracker® Deep Red. 235
MitoTrackers® are a class of cell permeable fluorescent probes which are based on a mildly thiol-236
reactive chloromethyl moiety specific for staining mitochondria. Briefly, cells after K-09 (IC50) 237
and CCCP treatment were harvested, washed once with PBS and immobilized to poly-l-lysine 238
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coated coverslips and were incubated with MitoTracker® Deep Red (100 nM, 20 min, RT) before 239
imaging. 240
241
2.13 Detection of cytochrome c release from mitochondria by immunofluorescence 242
microscopy 243
Cytochrome c release occurs after the opening of the mitochondrial permeability transition pore 244
(MPTP) and activates the downstream effectors such as the initiator caspases. Microscopic 245
localization of cytochrome c was performed as described previously (11). Briefly, cells with or 246
without drug treatments were fixed in 4% PFA, permeabilized with 0.1% Triton-X100 and 247
adhered to coverslips. Afterwards, cells were incubated with anti-cytochrome c primary antibody 248
(#sc-7159, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4°C and subsequently with 249
FITC-labeled secondary antibody (#GX-5011FC3R, Genetix Biotech, New Delhi, India) for 2h at 250
RT and processed for confocal imaging. 251
252
2.14 Measurement of intracellular ATP levels 253
Depletion of intracellular ATP pool following drug treatment was monitored by employing the 254
firefly luciferase bioluminescence based ATP detection assay (ATP Determination Kit, 255
Invitrogen) (17). Briefly, VC and K-09 treated cells (107/ml) were harvested by pelleting and 256
resuspended with boiling distilled water and were further boiled for 5 min for lysis. The 257
supernatant was added to the reaction mixture (1:19) (1 mM DTT, 0.5 mM D-Luciferin, 20× 258
Assay buffer, 1.25 µg/mL firefly luciferase), incubated for 10 min at 28°C and then read in a Bio-259
Tek FLx 800T multimeter (Bio-Tek, Winooski, VT, USA). 260
Oli-A (10 µM) treatment (till harvesting of cells) in promastigotes and amastigotes was given 2h 261
post-K-09 treatment in presence of low (1 mg/mL) and high glucose (4.5 mg/mL) media. Whether 262
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addition of oli-A post-K-09 exposure increased cell viability in Leishmania promastigotes in 263
presence or absence of glycolytic substrate glucose was measured using the CCK-8 cell counting 264
kit (Dojindo Laboratories, Kumamoto, Japan). 265
266
2.15 Imaging and quantification of cytoplasmic lipid droplets 267
Altered lipid metabolism during periods of stress might affect the quantity of lipid storage bodies 268
(lipid droplets or LDs) inside the cell, which would be revealed by quantifying and comparing 269
intracellular LDs pre- and post-treatment. The fluorescent lysochrome dye Nile Red (NR) which 270
selectively stains intracellular lipid droplets was used for this study (18). Briefly, VC and K-09 271
treated cells were harvested, washed once with PBS and stained with NR (1 μg/mL) for 10 min at 272
RT, followed by immobilization on poly-L-lysine coated coverslip and visualization under 273
confocal microscope. Mean number of LDs were calculated from ≥20 cells. 274
275
2.16 Detection of caspase-3/7 activity 276
A major feature of apoptotic death is the involvement of cysteine aspartate proteases (caspases) 277
that mediate events downstream of the mitochondria (19). Activity of caspases -3 and -7, two 278
major executioner caspases, was monitored following treatment with the NucView 488 Caspase-3 279
Assay Kit (Biotium, Hayward, CA, USA) according to manufacturer protocol. The assay uses an 280
inactivated DNA dye excited by 488nm laser, tagged with the tetrapeptide caspase-3 recognition 281
sequence DEVD (Asp-Glu-Val-Asp). Upon cleavage by caspase-3 (or its homologues) the 282
NucView substrate binds to cellular DNA and yields the active green fluorescent product; 283
unbound dye is non-fluorescent and is washed away. Cells treated or not with K-09 (IC50) and 284
with or without the caspase-3/-7 inhibitor Ac-DEVD-CHO (10 µM; 2h pre-treatment) were used 285
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in the experiment. Briefly after harvesting, cells were incubated with NucView488 substrate (5 286
µM, 30 min, RT) and thereafter immobilized on poly-L-lysine coated coverslips for imaging. 287
288
2.17 Detection of phosphatidylserine reversal by microscopy 289
Reversal of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane is 290
a characteristic early feature of apoptosis in Leishmania (12). To test this, we used FITC-tagged 291
Annexin-V, a protein that binds to externalized phosphatidylserine. The experiment was 292
performed according to manufacturer protocol (ApoDetect Annexin V-FITC kit, Invitrogen). Cells 293
treated or not with K-09 (IC50) dose and cells pre-incubated with caspase inhibitor Ac-DEVD-294
CHO (10 µM, 2h pre-treatment) were harvested, washed and resuspended in 1× binding buffer. 295
Annexin V FITC antibody mix was added and incubated for 10 min at RT. Cells were then 296
washed once with binding buffer, adhered to poly-L-lysine coated coverslips and processed for 297
confocal imaging. 298
299
2.18 Cell cycle analysis by flow cytometry 300
DNA content analysis was performed using propidium iodide (PI). Briefly, cells with and without 301
treatment were harvested, fixed in 70% ethanol (1h, 4oC). Afterwards the cells were pelleted, 302
washed with PBS and then resuspended in 300 µL of PBS. Then 35 µL of RNase A (10 mg/mL 303
stock) was added to the cells and kept at 37ºC for 30 min. Subsequently, 10 µl of PI (1 mg/mL) 304
stock was added to the samples which were then incubated on ice for 15 min and finally analyzed 305
by flow cytometry. 306
307
2.19 Detection of DNA fragmentation by TUNEL assay 308
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Cell death pathways involve the activation of different endonucleases, which cleave genomic 309
DNA into oligonucleosomal fragments of 180-200 bp (20). Terminal Deoxynucleotidyl 310
Transferase mediated dUTP Nick End Labeling (TUNEL) assay works by attaching a 311
Fluorescein-conjugated dUTP at the 3’-end of nicked DNA by the enzyme terminal 312
deoxynucleotidyl transferase (TdTase). The experiment was performed according to 313
manufacturers’ protocol (DeadEnd Fluorometric TUNEL System, Promega, Madison, WI, USA). 314
Cells were harvested, adhered on coverslips, fixed with 4% PFA and subsequently permeabilized 315
with 0.2% Triton X-100. After equilibrating the cells with TdT Equilibration Buffer, they were 316
incubated in the rTDT reaction mix containing equilibration buffer, nucleotide mix and rTdT 317
enzyme for 60 min at 37°C. Post-incubation, cells were washed once with 2×SSC and 318
counterstained with 10 µg/mL PI and visualized under confocal microscope. Another set of 319
samples were incubated in suspension in microcentrifuge tubes, following the same protocol as 320
for confocal imaging, and were analyzed by flow cytometry with a standard fluorescein/PE filter 321
set. 322
323
2.20 Statistical calculations 324
All experiments were performed in triplicate. Values stated are mean ± standard error of mean 325
(SEM). Statistically significant differences between two groups were calculated with unpaired 326
Student’s t-test; differences being significant when p<0.05. All calculations of fluorescence 327
intensity were performed on ≥20 cells in the field(s) that were displaying fluorescent signals. 328
Values, not otherwise stated, are mean of three or more similar experiments. 329
330
Results 331
3.1 K-09 induces morphological alterations in L. donovani promastigotes 332
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Scanning Electron Microscopy (SEM) was employed to assess morphological alterations in 333
Leishmania promastigotes induced by K-09. SEM analysis of control cells revealed healthy 334
parasites with typical slender bodies, long flagella and smooth cell surfaces. At IC50 dose, K-09 335
treated cells revealed striking morphological alterations. Cells had shrunk in volume and had 336
assumed round bag-like swollen appearance [Fig. 1(c-f)] with stumpy flagella. The cell surface 337
showed wrinkling and multi-septations were observed along the length of the cell [Fig. 1(d)]. 338
Such septations were not observed with miltefosine (standard anti-leishmanial drug) treated cells. 339
At 2×IC50 dose, cells were in the late stage of cell death and SEM micrographs showed that most 340
cells were distorted, lost their flagella completely and some of them lysed. A comparative 341
quantitative morphological analysis from SEM data is shown in Figure 1(i). 342
343
3.2 K-09 induces gross ultra-structural alterations typical of apoptosis 344
The subcellular alterations of the parasites induced by K-09 were analyzed by TEM thin 345
sectioning technique. Normal ultrastructure was observed in control promastigotes. Cells were 346
slender and elongated with kinetoplast containing highly condensed DNA. VC cells exhibited a 347
single ramified mitochondrion containing well defined cristae and electron-dense matrix which 348
extended throughout the length of the parasite [Fig. 2(i)]. Nucleus contained evenly distributed 349
chromatin. Cells treated at IC50 dose exhibited several ultra-structural distortions typical of 350
apoptotic cells. Reduction in size of the cells from slender elongated morphology was observed. 351
The cytoplasm appeared less electron-dense with increased cytoplasmic vacuolation, increased 352
accumulation of lipid droplets and multi-vesicular bodies [Fig. 2(e, f)]. Significant swelling and 353
disruption of kinetoplast-mitochondria complex were noticed with de-condensation of the kDNA. 354
Displacement of the kinetoplast and flagellar pocket disruption was also a common feature 355
noticed in treated parasites [Fig. 2(h)]. The affected mitochondria showed considerable swelling 356
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with disorganized cristae and loss in matrix density [Fig. 2(j)]. Considerable amount of pyknosis 357
in nuclei was observed while the plasma membrane remained intact. 358
359
3.3 K-09 depolarizes the mitochondrion triggering release of cytochrome c into the cytosol 360
Considering TEM observations on the disruptive effect of K-09 on the mitochondrion of the 361
parasites, we next assessed its effect on mitochondrial transmembrane potential (ΔΨm) using JC-362
1 staining. Confocal microscopy was used to visualize depolarization, along with flow cytometry 363
and fluorescence spectrophotometry to quantitate ΔΨm. Microscopic observations [Fig. 3(b)] 364
revealed that K-09 (IC50) treated cells exhibited significantly greater fluorescence in green region 365
indicating lower ΔΨm than VC cells which exhibited mitochondrial fluorescence in red region 366
indicating higher ΔΨm. K-09 treatment resulted in ~5 fold decrease in JC-1 fluorescence intensity 367
ratio (red/green). Similar response was observed in CCCP treated cells indicating depolarization 368
of the mitochondrial membrane. Gradual mitochondrial depolarization by K-09 was also 369
monitored by kinetic fluorometric measurements at 530 nm [Fig. 3(c)]. K-09 treatment (2×IC50) 370
resulted in ~2 fold increase in fluorescence intensity over VC cells after 90 min. Flow cytometry 371
analysis of K-09 treated cells at IC50 and 2×IC50 for 24h showed 87.5% and 97% depolarized 372
mitochondria respectively as against 9.5% in VC [Fig.3(g)]. Translocation of cytochrome c from 373
mitochondria into the cytosol after K-09 treatment was confirmed by immunofluorescence 374
microscopy [Fig. 3(d)]. 375
376
3.4 K-09 treatment impairs ATP production 377
Since intracellular ATP content is a direct marker of the energy state and thus the health of the 378
mitochondrion of the cell, we measured ATP content using a bioluminescence assay. K-09 379
treatment resulted in marked reduction in ATP levels in promastigotes as well as intracellular 380
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amastigotes. Total ATP content compared to VC was 4.2 and 4.8 fold lower in IC50 and 2×IC50 K-381
09 treated promastigotes respectively [Fig. 4(a)]. Addition of oli-A after 2h of IC50 K-09 exposure 382
in low glucose (LG; containing 1mg/mL glucose) and glucose free media caused severe depletion 383
in cellular ATP content. However, oli-A could not significantly prevent ATP depletion of K-09 384
treated cells in presence of high glucose (glycolytic substrate) [Fig. 4(c)]. In intracellular 385
amastigotes, we observed that addition of oli-A (in LG media) following K-09 treatment caused 386
4.2 fold drop in cellular ATP pool of the infected macrophages and depletion by 8.4 fold in the 387
presence of HG media when compared with infected macrophages treated with K-09 alone in HG 388
media [Fig. 4(d)]. 389
390
3.5 K-09 induced mitochondrial depolarization generates ROS and causes oxidative stress 391
Confocal microscopy of cells stained with the ROS probe carboxy-H2DCFDA revealed that 392
majority of the K-09 (IC50) treated cells fluoresced evenly throughout the cells while no 393
fluorescence was observed in VC cells indicating elevated ROS levels post-treatment [Fig. 5 (a)]. 394
Similar results were obtained with TBHP, a positive control. Cells pre-treated with NAC showed 395
fluorescence comparable to VC cells. After 24 h, K-09 treatment showed a 2.5 fold increase in 396
ROS levels as compared to VC [Fig. 5(d)] while NAC pre-treatment maintained ROS levels 397
almost at par with VC. Cell permeable fluorogenic probe MitoSOX™ Red was used as a 398
mitochondrial superoxide probe and visualized by confocal microscopy. Control cells exhibited 399
weak fluorescence whereas K-09 treated cells exhibited intense red fluorescence indicative of 400
severe oxidative stress [Fig. 5(b, c)]. To confirm whether mitochondrial depolarization precedes 401
ROS generation, we performed time lapse fluorometric experiments after pre-treating cells with 402
NAC and then exposing them to K-09. A steady increase in cytosolic calcium (using Fluo-4AM) 403
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and mitochondrial depolarization was observed but no increase in intra-cellular ROS level was 404
detectable [Fig. 5(f-h)]. 405
406
3.6 K-09 induced oxidative stress causes GSH depletion 407
Cells have developed the cellular thiol buffer system to neutralize free-radical damage, and its 408
principal component GSH (reduced glutathione) is an important free radical scavenger that works 409
by donating electron to ROS and thereby forming GSSH (oxidized glutathione) and thus aiding 410
the survival of the cell (11, 21). Cellular GSH levels in treated and NAC pre-treated cells were 411
monitored with MCB dye. We observed that NAC pre-treatment led to an increase in GSH 412
content of the cells over VC by salvaging free radicals, whereas IC50 and 2×IC50 doses of K-09 413
depleted cellular GSH levels by 45% and 33%, respectively [Fig. 5(e)]. 414
415
3.7 K-09 treatment results in elevation of cytosolic calcium levels 416
Since disruption in calcium homeostasis by its release from intracellular stores like ER and 417
acidocalcisomes is a critical event triggered by chemotherapeutic agents (15, 22), we explored the 418
effect of K-09 on the intracellular Ca2+ level using the dye Fluo-4AM, which has previously been 419
used to measure intracellular calcium (17). We observed prompt rise in the Fluo-4AM 420
fluorescence within 5 min after the addition of K-09 (2×IC50) which steadily increased following 421
K-09 treatment up to 1½h, after which it saturated to a plateau. At this point a ~2 fold increase in 422
fluorescence against VC was observed [Fig. 6(b)]. Ionomycin, a Ca2+ ionophore, rendered greater 423
than twice the fluorescence intensity of VC cells after 1½h while cells exposed to the drug in the 424
presence of Ca2+ chelator EGTA in the medium showed fluorescence similar to VC, indicating 425
that most of the calcium responsible for fluorescence increase is extracellular. These results are 426
corroborated by confocal microscopy findings. [Fig. 6(a, c)] 427
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428
3.8 K-09 induces Sub-G0/G1 phase cell cycle arrest in L. donovani promastigotes 429
DNA content analysis of untreated promastigote populations showed negligible cells in sub-G0/G1 430
region while cells treated with K-09 (IC50) showed about 57% cells as apoptotic in sub-G0/G1 431
region and cells treated with K-09 (2×IC50) showed 93% cells in the apoptotic fraction [Fig. 7]. 432
433
3.9 K-09 triggers phosphatidylserine reversal 434
Externalization of Phosphatidylserine (PS) was visualized using Annexin-V/FITC staining. We 435
observed that K-09 (IC50) treated cells displayed bright FITC fluorescence along their periphery 436
indicating PS externalization and induction of apoptosis [Fig. 8(a)]. In contrast, pre-treatment 437
with caspase-3/7 inhibitor Ac-DEVD-CHO led to inhibition of PS externalization after K-09 438
treatment. 439
440
3.10 Involvement of caspase-like proteases in K-09 induced apoptosis 441
To confirm caspase-like protease activity in K-09 mediated apoptosis, we employed confocal 442
imaging of a caspase-specific fluorescent substrate (NucView 488, Biotium). We observed 443
distinct fluorescence inside the nucleus of the cell in K-09 treated cells, a characteristic of this dye 444
where the caspase-cleaved fluorescent product binds to DNA. Such fluorescence was totally 445
absent in VC cells. Pre-treatment with Ac-DEVD-CHO before drug treatment prevented any 446
fluorescence in the nuclear region [Fig. 8(b)]. 447
448
3.11 DNA fragmentation on K-09 exposure 449
To investigate induction of DNA fragmentation on K-09 exposure, TUNEL assay was performed. 450
The samples were analyzed by flow cytometry and reveal approx. 4% TUNEL+ cells in untreated 451
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promastigotes and 58% and 86% of TUNEL positive cells in IC50 and 2×IC50 doses of K-09 452
respectively after 24h of drug treatment [Fig. 8(c)]. 453
454
3.12 K-09 treatment alters lipid metabolism in L. donovani promastigotes 455
Since TEM analysis revealed increased number of lipid droplets after K-09 treatment, we 456
confirmed this observation with Nile Red staining of live parasites. Significant increase in the 457
number of cytoplasmic lipid droplets at K-09 (IC50) [Fig. 8(d, e)] was observed. However, at 458
2×IC50, the smaller droplets probably fused to form larger ones, represented by a corresponding 459
decrease in mean droplet count. 460
461
Discussion 462
The promising anti-leishmanial agent K-09 was recently reported to be safe and orally active in 463
the hamster model for VL (13). This clerodane diterpene was shown to be a DNA topoisomerase 464
inhibitor using biochemical assays and molecular docking studies (13). The detailed mode of 465
action of this agent however remained uncharacterized. Therefore, in the present study, we have 466
investigated the physiological and ultra-structural effects of K-09 on L. donovani promastigotes to 467
further dissect the mechanism of cell death induction by this compound. 468
469
Morphological alterations in L. donovani promastigotes induced by K-09 were studied using 470
electron microscopy which still remains the gold standard for diagnosing nature of cell death (23). 471
Since the genus Leishmania diverged early in the evolution from the metazoans, there are 472
significant differences in cell death at molecular level in this unicellular organism. This makes it 473
difficult to interpret results when commonly used metazoan apoptotic biomarkers are used in cell 474
death assays. Unicellular kinetoplastid parasites have special organelles involved in essential 475
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metabolic pathways with steps differing from their mammalian counterparts thus making them 476
attractive targets for new chemotherapeutic agents. Here, ultra-structural studies can be very 477
helpful in achieving this goal (24). Our SEM observations revealed swelling and overall rounding 478
up of K-09 treated cells with significant loss in body length and shortening of flagella compared 479
to untreated cells. One notable finding was the presence of distinct membrane folds in K-09 480
treated cells. This feature was not observed in miltefosine treated cells. Similar observations have 481
also been reported in promastigotes upon treatment with geldanamycin and cyclosporine A (25, 482
26), where the molecular chaperone Hsp90 inactivation has been implicated. Probably, K-09 too 483
might be directly or indirectly inhibiting Hsp90 to trigger such topological alterations. TEM 484
observations revealed that K-09 treatment resulted in morphological features typical of apoptosis. 485
Sub-cellular alterations included rounding up of cells, intense cytoplasmic vacuolation, pyknosis 486
of nucleus, structural disruption of mitochondrion and decondensation of the kinetoplast. Previous 487
studies have described similar observations of mitochondrion-kinetoplast damage upon treatment 488
in L. amazonensis promastigotes with a putrescine analogue (27), antiarrhythmic drug amiodarone 489
(22), and a squalene synthase inhibitor BPQ-OH (28). 490
491
Since the mitochondrion plays a pivotal role in orchestrating apoptosis and these parasites possess 492
only one large ramified mitochondrion that caters to majority of their energy requirements (29), 493
irreversible damage and dysfunction of this vital organelle would have disastrous consequences 494
on their survival. Thus, these observations have serious implications for drug design against this 495
parasite. Disruption of the structural integrity of the mitochondria through damage to its inner 496
membrane might also account for de-condensation of the kinetoplast since inner membrane holds 497
the kinetoplast (30). Membrane blebbing, a common feature in metazoan apoptosis was notably 498
absent which is not surprising since this unicellular eukaryote has different set of molecular 499
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players controlling apoptosis when compared to metazoans. Alterations in the flagellar pocket 500
region without major plasma membrane disruption at IC50 dose was another major evidence 501
suggesting disruption of microtubule dynamics leading to inhibition of intracellular trafficking of 502
nutrients by K-09 since endo/exocytosis are the central processes which take place in this cellular 503
region (14, 31). 504
505
Although TEM micrographs identified mitochondrion as the severely affected organelle upon K-506
09 exposure, the underlying mechanism of action still remained elusive. Thus we investigated 507
whether K-09 affected mitochondrial membrane potential and processes driven by it such as ATP 508
production, especially since the parasite depends mainly on oxidative phosphorylation for ATP 509
production (29). Our observations point out that K-09 depolarized the parasite mitochondria and 510
impaired ATP production. We also sought to understand whether oligomycin-A (inhibitor of the 511
mitochondrial F0/F1-ATP synthase), which is known to prevent ATP depletion and rescue 512
procyclic Trypanosoma brucei cells from cell death in glucose-rich conditions, could do the same 513
in K-09 treated Leishmania donovani. Oli-A treatment alone prevented ATP depletion in presence 514
of high glucose compared to glucose-free media but not in K-09 treated cells. This observation is 515
in agreement with previous studies that report the existence of an energy compensatory 516
mechanism in the form of substrate level phosphorylation that is activated in procyclic form of T. 517
brucei under conditions that favor glycolysis or when oxidative phosphorylation is under stress by 518
agents such as oli-A (32). Additionally, oli-A in HG media post-K-09 exposure could not rescue 519
cells when compared with oli-A in LG media. A possible explanation could be that mitochondrial 520
depolarization, a so-called point-of-no-return of cell death, may be an upstream event of ATP 521
depletion in K-09 induced apoptosis and cells with depolarized mitochondria may be irreversibly 522
committed to the cell death pathway. The release of pro-apoptotic protein cytochrome c, an 523
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essential component of the electron transport chain that localizes in mitochondria, into the cytosol 524
of the parasite upon mitochondrial dysfunction is another characteristic feature of PCD in 525
kinetoplastids (19, 33). The release of cytochrome c into cytosol post– K-09 treatment suggests 526
mitochondrial PTP opening as another important event initiated by this diterpene. 527
528
While mitochondrial depolarization and cytochrome c release are critical, it is the subsequent 529
elevation in the levels of oxidizing species generated from mitochondria that plays the role of 530
cytotoxic effectors in apoptosis. In Leishmania, as in other metazoans, there is a basal level of 531
ROS maintained by the mitochondrion inside the cells for physiological signaling (34). The onset 532
of mitochondrial dysfunction due to disruption of its structural integrity causes leakage in the 533
ETC and thus elevates ROS levels. Reports of anti-trypanosomal activity for mitochondrial 534
disruptors include 3,3′-diindolylmethane (17), tafenoquine (35), and sitamaquine (36) which 535
inhibit F0-F1 ATP synthase, complex III and complex II of the parasite mitochondrion 536
respectively. Anti-leishmanial agents such as baicalein (33), amiodarone (22) induce oxidative 537
stress and cytosolic calcium increase respectively, which then depolarizes mitochondrion to 538
generate ROS. Elevated ROS levels are buffered by cellular GSH pool which causes its depletion 539
(17, 37). Dose-dependent depletion of cellular GSH pool and increase in ROS levels on K-09 540
exposure were observed while pre-treatment with NAC maintained the GSH pool and ROS levels 541
at par with VC cells. NAC pre-treatment prior to K-09 exposure however was unable to prevent 542
mitochondrial depolarization and intracellular calcium rise. This demonstrates that intra-cellular 543
ROS generation occurs downstream of mitochondrial depolarization and intracellular calcium 544
rise. 545
546
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Disruption of intracellular calcium homeostasis is another characteristic feature of PCD in 547
Leishmania. Several studies have reported mitochondrial depolarization and subsequent ROS 548
generation to be intimately associated with dysregulation of intracellular calcium homeostasis 549
(10, 15, 22, 33). K-09 treatment results in elevation in cytoplasmic calcium possibly due to the 550
influx of Ca2+ from extracellular medium through the activation of plasma membrane Ca2+-551
ATPase (PMCA). 552
553
K-09 treatment also resulted in increase in the number of lipid droplets inside the promastigote 554
which was observed by TEM analysis and NR staining. Similar observations have been reported 555
in L. amazonensis after treatment with squalene synthase inhibitors and amiodarone (31, 22). 556
These observations were attributed to inhibition of sterol biosynthesis in the parasite causing 557
formation of lipid bodies composed of abnormal intermediate metabolites (31). Interestingly, 558
previous studies have implicated this diterpene as an inhibitor of HMG-CoA reductase (HMGR) 559
(38), a rate-limiting enzyme present in the mitochondria of trypanosomatids and required for the 560
synthesis of ergosterols (39). Also a study on the regulation of HMGR of L. major reported that 561
the enzyme has higher affinity for lovastatin, its competitive inhibitor than its natural substrate 562
HMG-CoA. In another study, incubation with lovastatin however activated an increased HMGR 563
activity in the promastigotes (40). It may be probable that the rise in the number of lipid droplets 564
observed on K-09 treatment might be due to the hyperactivity of HMGR, as K-09 was shown to 565
be structural analog of lovastatin (38). This presumably leads to overproduction of the lipid 566
precursors that accumulate in the form of lipid droplets. 567
568
It is well known that caspases, a family of cysteine proteases are involved in orchestrating 569
apoptosis in metazoans. Caspase holomogues known as metacaspases in Trypanosoma and 570
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Leishmania have been reported to play distinct roles in PCD (41, 42), though their caspase-571
substrate cleaving activity has been debated, as is the role of the requirement of an active site 572
cysteine in the substrate (42). However, the Ld caspase homologues LdMC1 and LdMC2 have 573
been shown to be essential in cell cycle proliferation (43). Studies have detected significantly 574
higher amount of active-form metacaspase in cells undergoing H2O2 induced PCD (10), and 575
elevated metacaspase gene expression in miltefosine induced PCD (44). However, studies that 576
report caspase-independent PCD in these parasites might suggest that metacaspase involvement 577
may not be essential (15, 36). In our study, we found major up-regulation of caspase-3/7 -like 578
protease levels in cells following K-09 exposure and pretreatment of cells with caspase-3 inhibitor 579
Ac-DEVD-CHO diminished caspase-3/7-like protease activity as well as PS externalization. 580
581
After its identification and first report from P. longifolia in 1988 (45), K-09 has been reported to 582
show diverse pharmacological activities such as antimalarial (46), antimicrobial and 583
antidyslipidemic (47, 38), before being recently reported as an anti-leishmanial by topoisomerase 584
I inhibition (13). In summary, we have shown that K-09 induces PCD in L. donovani 585
promastigotes by mitochondrial dysfunction causing elevation of ROS to cytotoxic levels inside 586
the cells that mediates cell death in a caspase-like protease-dependent manner. Our study adds to 587
the growing body of evidence of the presence of an apoptosis-like PCD mechanism in these 588
unicellular protists similar to metazoan PCD. Identification and characterization of molecular 589
players involved at different checkpoints can be useful in discovering and screening potential 590
molecular targets for rational drug design. In conclusion, our work provides a basis for further 591
investigation towards development of K-09 as a potential drug candidate for VL. 592
593
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Acknowledgements 594
The authors thank Dr. A.A. Sahasrabuddhe for access to his cell culture laboratory during the 595
initial stages of the study and for sharing L. donovani strain MHOM/IN/80/DD8. Mr. A.L. 596
Vishwakarma, is acknowledged for flow cytometry analysis and Dr. (Mrs.) K. Singh and Mrs. M. 597
Srivastava for technical assistance during TEM sample preparation. The authors thank Dr. N. 598
Goyal for sharing the J774.A1 macrophage cell line, Drs. R.S. Bhatta and D.P. Mishra for access 599
to fluorescence spectrophotometers. This is CDRI Communication No. 149/2013/KM. 600
601
Funding 602
Funding from Council of Scientific and Industrial Research (CSIR) network project BSC0114 is 603
gratefully acknowledged. MK and AB are recipients of CSIR JRF. 604
605
Conflicts of Interests 606
The authors declare no conflict(s) of interests. 607
608
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causes cell-cycle defects and programmed cell death in Leishmania donovani. Mol. Cell 734
Biochem. 359: 135–49. 735
44. Khademvatan S, Gharavi, MJ, Saki J. 2011. Miltefosine induces metacaspase and 736
PARP genes expression in Leishmania infantum. Braz. J. Infect. Dis. 15: 442–8. 737
45. Phadnis AP, Patwardhan SA, Dhaneswar NN, Tavale SS, Guru TN. 1988. Clerodane 738
diterpenoids from Polyalthia longifolia. Phytochemistry. 27:2899-2901. 739
46. Ichino C, Soonthornchareonnon N, Chuakul W, Kiyohara H, Ishiyama A, Sekiguchi 740
H, Namatame M, Otoguro K, Omura S, Yamada H. 2006. Screening of Thai medicinal 741
plant extracts and their active constituents for In Vitro antimalarial activity. Phytother. 742
Res. 20:307-309. 743
47. Murthy MM, Subramanyam M, Bindu MH, Annapurna J. 2005. Antimicrobial 744
activity of clerodane diterpenoids from Polyalthia longifolia seeds. Fitoterapia. 76:336-745
339. 746
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Figure Legends: 748 749
Fig. 1. SEM micrographs showing altered morphology of L. donovani promastigotes in K-09 750
treated (c-f) and miltefosine treated (g-h) cells compared to VC (a-b). Note the round cell bodies 751
(c-f), shortened flagella (c-f) and presence of membrane folds (c-d) compared to slender body 752
with long flagella in VC cells (a-b); (i) Mean body and flagellar length in VC and K-09 (IC50 and 753
2×IC50) treated parasites. Bars=1µm; ***p<0.05 754
755 756
Fig. 2: Ultra-structural alterations following K-09 administration in L. donovani promastigotes. 757
Cells treated with IC50 (b, e and h) and 2×IC50 (f and j) doses of K-09 show dramatic alterations in 758
ultra-structure compared to VC cells (a, d, g and i). Note the change in overall morphology, 759
nuclear condensation (e), distortion of the flagellar pocket (h), appearance of lipid reservoirs 760
(asterisk in e) and multi-lamellar bodies(MB) (f), fragmentation of the mitochondrion 761
(arrowheads in e, h) and disruption of the mitochondria-kinetoplast complex (asterisk in f) with 762
disorganized cristae (arrowhead in j) against VC cells (inset in i). Also note the increase in 763
vacuoles (star in e) and acidocalcisomes (black arrows in e). N=nucleus, K=kinetoplast, 764
F=flagellum, FP=flagellar pocket, M=mitochondrion, MB=multilamellar bodies. Bars=1µm (a-f), 765
500nm (g-j); inset=250nm. 766
767
Fig. 3: Depolarization of the L. donovani mitochondrion and release of cytochrome c into the 768
cytosol following K-09 treatment. (a) Pattern of mitochondrial distribution along the cell before 769
and after treatment with K-09 and CCCP, note the ramified mitochondrial network present in VC 770
cells is disrupted upon K-09 and CCCP treatment. (b) Confocal imaging of cells treated with K-771
09 and stained with JC-1, showing increased fluorescence in the green channel indicating 772
depolarized mitochondria. (c) Gradual mitochondrial depolarization in cells treated with K-09, 773
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CCCP and stained with JC-1 showing sharp fall in ΔΨm, indicated by increasing JC-1 774
fluorescence in green channel. (d) Confocal images of VC and cells treated with K-09 and stained 775
for cytochrome c localization. Note the same ramified pattern of mitochondrion from (a) in VC, 776
and the diffused cytosolic staining in treated cells, indicating cytochrome c release from 777
mitochondria. (e) Ratio of fluorescence (red/green channel) from (b), showing ΔΨm loss upon K-778
09 treatment. (f) Ratio of fluorescence intensity in red/green channel after 24h treatment. (g) Flow 779
cytometric analysis of K-09 and CCCP treated cells showing similar results. Bars=5µm; 780
***p<0.001. 781
782
Fig. 4: Depletion of cellular ATP pool in L. donovani promastigotes and amastigotes post K-09 783
treatment. (a) Intracellular ATP levels decrease with K-09 and CCCP. (b) Addition of oli-A in 784
presence of glucose prevented ATP depletion in promastigotes, but not in case of K-09 treated 785
cells. (c) Survival of promastigotes was not significantly altered with the addition of oli-A in K-786
09 treated cells in low glucose (Low gluc) or high glucose (Hi gluc). (d) K-09 also induced ATP 787
depletion in amastigotes, which was moderately reduced by the addition of oli-A in presence of 788
either low or high glucose. HG=high glucose, LG= low glucose, -G=no glucose. p*<0.05, 789
p**<0.01, p***<0.001, n.s. p>0.05, n.s. = non-significant 790
791
Fig.5: K-09 generates reactive oxygen species (ROS) and promotes oxidative stress. (a) Reactive 792
oxygen species levels visualized with CM-H2DCFDA and analyzed by confocal microscopy, note 793
that NAC prevents increment of ROS levels, (b) Confocal micrograph of cells stained with 794
MitoSOX™ Red, (c) percentage of cells showing MitoSOX™ Red fluorescence, analyzed by 795
flow cytometry, (d) CM-H2DCFDA fluorescence post-24h K-09 treatment, (e) Cellular reduced 796
glutathione levels post-24h treatment with MCB staining. Monitoring changes in (f) intracellular 797
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ROS, (g) mitochondrial membrane potential and (h) Ca2+ levels when cells were pretreated with 798
NAC (20 mM, 2h). Bars=5µm. 799
800
Fig. 6. Increment of intracellular Ca2+ levels following K-09 administration. (a) Cells stained with 801
the Ca2+ sensor dye Fluo-4AM and analyzed by confocal microscopy. Note that presence of Ca2+ 802
chelator EGTA during K-09 treatment abrogates the rise of Ca2+ levels. (b) Intracellular Ca2+ 803
levels following K-09 administration monitored up to 90min. (c) Fluo-4 AM fluorescence post-804
24h treatment with K-09 measured using fluorometer. 805
806
Fig.7: Cell cycle arrest in L. donovani promastigotes on K-09 treatment. DNA content analysis 807
post 24h of drug treatment was performed after PI staining using flow cytometry. Note the normal 808
cell cycle profile of VC cells while K-09 treatment arrests cells in sub-G0/G1 phase in proportion 809
to the dose of K-09 IC50 and 2×IC50. 810
811
Fig. 8: Apoptotic markers in K-09 treated parasites. (a) Annexin-V/FITC stained cells with and 812
without K-09(IC50) treatment. VC cells show no Annexin-V/FITC fluorescence while K-09 813
treated cells show annular fluorescence along the cell periphery, suggesting reversal of PS. Cells 814
pre-treated with the caspase-3/7 inhibitor Ac-DEVD-CHO show decrease in fluorescence, 815
implying that apoptosis has been prevented. (b) Caspase-3/7 like protease activity present in 816
apoptosis following K-09 treatment, which is abrogated by Ac-DEVD-CHO. (c) Flow cytometric 817
analysis of TUNEL+ cells after K-09(IC50 and 2×IC50) treatment. Confocal imaging (d) and 818
quantification of number of lipid droplets (e) post- K-09 treatment (single-plane image). 819
Bars=5µm, ***P<0.001. 820
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