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1 Department of Infectious Disease and Immunology, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata 700032, India
2 Steroid and Terpenoid Chemistry, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata 700032, India
Correspondence
Chitra Mandal
cmandal{at}iicb.res.in
Received 8 December 2006
Accepted 7 May 2007
Abbreviations: MFI, mean fluorescence intensity; PCD, programmed cell death; PI, propidium iodide; SAG, sodium antimony gluconate; TdT, terminal deoxynucleotidyltransferase; TUNEL, TdT-mediated dUTP end labelling.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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Programmed cell death (PCD) mechanisms are known to be operative in kinetoplastid parasites of the genus Trypanosoma (Szallies et al., 2002) and Leishmania (Arnoult et al., 2002) in response to various chemotherapeutic stimuli such as pentostam, amphotericin B (Lee et al., 2002) and miltefosine (Paris et al., 2004).
Plant-derived products have shown promise in the search for better therapeutics against leishmaniasis (Dupouy-Camet, 2004). In this regard, the leaf exudate of Aloe vera (Dutta et al., 2007a, b) and triterpenoid saponin of Careya arborea (Mandal et al., 2006a) have been reported to be leishmanicidal. Although water-insoluble saponins have shown anti-leishmanial properties (Delmas et al., 2000; Maes et al., 2004), to our knowledge, no leishmanicidal activity mediated by PCD has been demonstrated.
The main objectives of the present study were to evaluate the anti-leishmanial effect and putative mechanism(s) of a water-soluble steroidal saponin, racemoside A, purified from the fruits of Asparagus racemosus. Here, we report, for the first time, that racemoside A is a potent anti-leishmanial molecule against both promastigotes and amastigotes of Leishmania donovani, acting by inducing PCD.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Parasite culture. Promastigotes were obtained from two Indian L. donovani strains, AG83 (MHOM/IN/83/AG83) and GE1F8R (a subclone of MHOM/IN/90/GE1) (Dutta et al., 2005). Based on their clinical response to sodium antimony gluconate (SAG), they were classified as SAG sensitive (AG83) or SAG resistant (GE1F8R). They were routinely cultured at 22 °C in complete medium (M199 medium supplemented with 10 % heat-inactivated FCS and 100 µg gentamicin ml–1).
Extraction, isolation and structure elucidation of racemoside A.
Racemoside A was purified from dried fruits of Asparagus racemosus (Mandal et al., 2006b). The compound was characterized as (25S)-5ß-spirostan-3ß-ol-3-O-{ß-D-glucopyranosyl(1
6)-[
-L-rhamnopyranosyl(16)-ß-D-glucopyranosyl(14)]-ß-D-glucopyranoside} with the molecular formula C51H84O22 (Fig. 1
). Racemoside A was dissolved in RPMI 1640 at a concentration of 1 mg ml–1 for evaluation of its anti-leishmanial activity.
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Anti-leishmanial activity in a macrophage–amastigote model (ex vivo). Murine peritoneal macrophages were lavaged using cold medium A from the peritoneum of BALB/c mice following starch induction (2 % starch administered intraperitoneally, 2 ml per mouse) and harvested in cold medium A. Cells (5x105) were then seeded on 16-well tissue culture glass slides (0.10 ml per well) and allowed to adhere overnight in an atmosphere of 5 % CO2 at 37 °C. After removal of non-adherent macrophages, stationary-phase promastigotes of strain AG83 were added to the macrophages at a ratio of 5 : 1 (parasite : macrophage) and incubated overnight in the same environment. Non-phagocytosed parasites were removed by gentle washing and infected macrophages were incubated with racemoside A (0–1 µg ml–1) or amphotericin B (0–1 µg ml–1) for an additional 72 h. The slides were fixed in methanol and Giemsa stained, and the number of amastigotes within macrophages was determined microscopically. The number of parasites per 100 macrophages per well was determined from quadruplicate cultures and the IC50 was established from the number of amastigotes in 100 macrophages. The ex vivo effect of racemoside A on the antimonial-resistant strain GE1F8R was enumerated in a similar way.
In parallel, the toxic effect of racemoside A on murine peritoneal macrophages was determined by incubating cells for 72 h in the presence or absence of racemoside A (0–10 µg ml–1) in medium A and evaluating the viability of macrophages as above. The absorbance by macrophages in the absence of racemoside A was considered to be 100 %.
Analysis of promastigote cellular morphology. Morphological changes in parasites as a result of racemoside A treatment were identified microscopically. Briefly, exponential-phase promastigotes were incubated in the absence (controls) or presence of racemoside A (10 µg ml–1) for 0–24 h in medium A. At various time points, cells were centrifuged, fixed in 4 % paraformaldehyde, attached to poly-L-lysine-coated glass slides, mounted in glycerol and examined under a confocal microscope (Leica). At least 20 microscope fields were observed for each sample.
Analysis of externalized phosphatidylserine in promastigotes by flow cytometry. Exponential-phase promastigotes were incubated with racemoside A (10 µg ml–1) for 0–6 h. Cells were centrifuged (3000 g for 5 min), washed twice in PBS and resuspended in annexin 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 according to the manufacturers' instructions, and incubated for 30 min in the dark at 20–25 °C. Data acquisition was carried out on a FACSCalibur flow cytometer (BD) and analysed using CELLQUEST software. If there is an alteration of the membrane integrity (due to externalization of phosphatidylserine), annexin V detects both pro- and late-apoptotic cells. Therefore, the simultaneous addition of PI, which does not enter healthy cells with an intact plasma membrane, discriminates between pro-apoptotic (annexin 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.
Measurement of the mitochondrial membrane potential of promastigotes.
Mitochondrial damage was assessed using a cell-permeable dye, JC-1, to analyse the change in mitochondrial transmembrane potential (
m). The carbocyanine dye JC-1 exists as a monomer or as aggregates, depending on concentration and membrane potential. In the mitochondria of healthy cells, with high m, the dye spontaneously forms complexes, known as J-aggregates, that emit red fluorescence at 585 nm. Following the onset of apoptosis, as the mitochondrial potential decreases, JC-1 remains in the monomeric form, emitting green fluorescence at 530 nm. Thus, the ratio of red (585 nm) to green (530 nm) fluorescence represents m, which is dependent only on the membrane potential (Reers et al., 1995). After exposure to racemoside A (10 µg ml–1) for 0–6 h, exponential-phase promastigotes were centrifuged, resuspended in PBS containing JC-1 (10 µg in 0.1 ml per well) and incubated at 37 °C for 10 min. Analysis of the mean green and red fluorescence intensities was carried out using a FACSCalibur and CELLQUEST software. The mitochondrial transmembrane potential was calculated from the ratio of the mean fluorescence intensity (MFI) of J-aggregates and monomers.
Analysis of the cell cycle. Exponential-phase promastigotes were incubated for 0–24 h in the presence of racemoside A (10 µg ml–1) and then washed twice with PBS (pH 7.2). Cells were fixed in chilled methanol by incubating for 3 min on ice and then resuspended in 0.5 ml PBS containing 0.5 µg PI and 50 µg RNase A and incubated for 1 h in the dark at room temperature. Data acquisition was carried out using a FACSCalibur and analysed using CELLQUEST software.
In situ detection of DNA fragmentation of promastigotes. DNA fragmentation within the cell was analysed by terminal deoxynucleotidyltransferase (TdT)-mediated dUTP end labelling (TUNEL) using an Apo-Direct kit according to the manufacturer's instructions. Briefly, promastigotes were incubated with racemoside A (10 µg ml–1 for 0, 18 or 24 h), washed twice in PBS (pH 7.2) and fixed in 1 % paraformaldehyde for 30 min. Cells were washed again in PBS and incubated in chilled 70 % ethanol for 30 min on ice. After washing, these cells were allowed to react with TdT enzyme in reaction buffer and stained with PI and dUTP–FITC. Finally, cells were resuspended in PBS before data acquisition using a FACSCalibur and CELLQUEST software.
Statistical analysis. In vitro anti-leishmanial activity was expressed as IC50 values by linear regression analysis. Values were determined as means±SD from at least three independent experiments in duplicate. P values were estimated using Student's t-test.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The established anti-leishmanial drug amphotericin B, used as a positive control, showed a similar trend in dose-dependent parasite killing, with IC50 values of 0.17 and 0.22 µg ml–1 for AG83 and GE1F8R, respectively.
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100-fold higher than that required for racemoside A (10 µg ml–1). Anti-leishmanial activity of a water-insoluble triterpenoid saponin isolated from C. arborea has been reported against L. donovani promastigotes, with an IC50 of 15 µg ml–1 (Mandal et al., 2006a), but its effect against the intracellular form of the parasite (amastigotes) has yet to be elucidated. Similarly, water-insoluble sapogenin from Asparagus africanus was parasiticidal (IC50 31±4 µg ml–1) against Leishmania major promastigotes (Oketch-Rabah et al., 1997). However, this sapogenin has not been tested against VL-causing forms of Leishmania such as L. donovani, which cause the most fatalities. Racemoside A has sugar moieties attached to the core structure and this core structure is similar to the structure of sapogenin. Our data demonstrated that racemoside A, a unique water-soluble steroidal saponin, is much more active against L. donovani promastigotes (IC50 1.15–1.31 µg ml–1; Fig. 2a
) than is the water-insoluble sapogenin (IC50 31±4 µg ml–1). Therefore, it can be envisaged that these additional sugar molecules not only increase the compound's hydrophilicity, but also enhance its leishmanicidal efficacy. Triterpenoid saponins isolated from Maesa balansae showed anti-leishmanial efficacy against L. donovani and Leishmania infantum at very low doses, with IC50 values of 7–40 ng ml–1 (Germonprez et al., 2005; Maes et al., 2004). Unfortunately, unlike racemoside A, these triterpenoid saponins are water-insoluble. To our knowledge, this is the first report of the leishmanicidal efficacy of a naturally occurring, water-soluble, steroidal saponin.
Racemoside A-mediated death of L. donovani amastigotes
Treatment with racemoside A demonstrated a dose-dependent removal of phagocytosed amastigotes (Fig. 2b), as shown by microscopic observation of Giemsa-stained cells. The IC50 of racemoside A in amastigotes was 0.17 µg ml–1 in strain AG83 and 0.16 µg ml–1 in strain GE1F8R, which is comparable to the IC50 of amphotericin B, used as a reference compound, with an IC50 of 0.16 µg ml–1 against AG83. The cytotoxic effect of racemoside A against murine peritoneal macrophages was enumerated biochemically using an MTT assay. The viability of murine peritoneal macrophages was >92 % after treatment with racemoside A (1 µg ml–1; Fig. 2c). A similar degree of macrophage viability (>89 %) was observed up to a concentration of 10 µg racemoside A ml–1.
Racemoside A is thus highly potent at very low doses, even against the intracellular form of the parasite, i.e. approximately 7-fold more effective (IC50 0.16–0.17 µg ml–1) against amastigotes than against promastigotes, which was comparable with the effect of amphotericin B (IC50=0.16 µg ml–1; Fig. 2b). More importantly, the absence of shared toxicity towards murine peritoneal macrophages, even up to a concentration of 10 µg ml–1 (Fig. 2c), increases its therapeutic ratio (the ratio between leishmanicidal concentration and the concentration that is toxic to host cells). The efficacy of racemoside A against intra-macrophage amastigotes (IC50 0.17 µg ml–1) was found to be higher than that of known anti-leishmanial compounds such as Pentostam (IC50 4.9–50 µg ml–1), miltefosine (IC50 13.6 µM; Paris et al., 2004), SAG (IC50 154 µg ml–1; Roberts & Rainey, 1993) and lipid formulations of amphotericin B (IC50 0.2–2.6 µg ml–1; Yardley & Croft, 2000), but comparable to amphotericin B (IC50 0.013–0.18 µg ml–1; Yardley & Croft, 2000). Higher leishmanicidal efficacy (low IC50), solubility in water and less shared toxicity towards mammalian cells (even at 10 µg ml–1) make this readily available plant-derived compound a potent anti-leishmanial molecule for SAG-sensitive and -resistant strains and their amastigote forms, and thus it merits further pharmacological investigation.
Racemoside A causes changes in promastigote morphology
During PCD, the promastigotes alter to an ovoid shape with nuclear condensation, which is accompanied by the formation of fragmented nuclei (Sen et al., 2004). Phase-contrast microscopic studies on the morphology of racemoside A-treated L. donovani promastigotes demonstrated the induction of certain morphological changes that were similar to the signs of PCD. The flagellated promastigotes shrank and became aflagellated and oval or round with an increase in vacuoles as a result of racemoside A exposure. The nuclei of control promastigotes displayed a prominent central or slightly eccentrically localized nucleolus, whilst chromatin was usually distributed peripherally beneath the nuclear membrane (Fig. 3). However, in most of the treated cells, condensed, irregular, bead-like, marginated chromatin and a fragmented nucleus with condensed cytoplasm were observed (Fig. 3).
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Accordingly, in order to determine whether racemoside A triggered cell death following a similar phenomenon, promastigotes treated with racemoside A (10 µg ml–1 for 0–6 h) were double-stained with annexin V–FITC and PI. The percentage of racemoside A-treated promastigotes that were positive only for annexin V gradually decreased from 11.52 % after 30 min to <1.0 % after 1 h (Fig. 4). However, the number of cells that were both annexin V- and PI-positive (Fig. 4, upper-right quadrant) gradually increased, to 2.54±1.05, 47.01±2.07 and 76.13±0.89 % at 15 min, 30 min and 1 h, respectively, indicating late-apoptotic phase. A gradual decrease in these annexin V- and PI-positive cells was observed at 4 and 6 h of treatment, to 50.86±3.28 and 38.33±4.28 %, respectively, with a simultaneous increase in the percentage of necrotic cells (annexin V-negative and PI-positive; Fig. 4, upper-left quadrant), to 41.59±2.45 % and 52.59±2.08 %, respectively. This was due to degradation of the parasites. These observations suggested that racemoside A induced cell death by loss of membrane integrity, as shown by the increased PI incorporation and annexin V binding, indicating late-apoptotic phase. In contrast, only 0.75±0.08 % of untreated cells were annexin V- and PI-positive, and the number of cells positive for PI only was negligible at all time points (0.07±0.02 %).
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Racemoside A induces sustained depolarization of mitochondrial membrane potential
The mitochondrial membrane potential (m) is commonly determined by the ratio of red : green fluorescence at 585 and 530 nm, i.e. J-aggregates in mitochondria versus monomers in the cytosol, using JC-1 staining (Reers et al., 1995; Sen et al., 2004). Incubation of promastigotes with racemoside A (10 µg ml–1) for 3 h caused depolarization, shown by a 16.49 % decrease in the 585 : 530 ratio in cells incubated in the presence of racemoside A compared with unexposed cells (Fig. 5; 11.34±0.35 vs 13.58±0.39, P<0.05). By 4 h, the decrease was 18.25 % (Fig. 5; 11.11±0.51 vs 13.59±0.59) and this was sustained up to 5 h. A maximum decrease of 32.64 % was observed at 6 h (Fig. 5; 9.12±0.37 vs 13.54±0.53, P<0.02). Taken together, these results indicated that exposure to racemoside A caused sustained mitochondrial membrane depolarization for up to 6 h, which may be due to imperfect mitochondrial function.
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). The reduction in formazan formation served as a measure of the decrease in the number of viable cells and was also associated with a decrease in mitochondrial membrane potential, corroborated by the decreased red fluorescence of racemoside A-treated promastigotes following JC-1 staining (Fig. 5). This change in mitochondrial membrane potential appeared to occur more slowly than the PI/annexin V double staining at 3 h, when the majority of cells were already in the late-apoptotic phase. The cells are driven into the late-apoptotic phase by changes in the morphology of the cell membrane, which thus initiates the cascade of events involving the cell organelles (such as mitochondria) leading to PCD. Most probably, racemoside A directly targets the plasma membrane making it leaky and this in turn switches on the mitochondria-mediated death signal cascades. The depolarization is known to commit cells to DNA fragmentation (Sen et al., 2004).
Racemoside A causes cell-cycle arrest in L. donovani promastigotes
Treatment of promastigotes with racemoside A (10 µg ml–1) for 6, 18 and 24 h caused cell-cycle arrest at the sub-G0/G1 phase, as analysed by flow cytometry. The proportions of cells in this phase at these time points were 5.04, 8.06 and 15.33 % compared with 1.61, 0.65 and 1.58 %, respectively, for cells that were not exposed to racemoside A (Fig. 6). This suggests that a check in the cell cycle could initiate late events of PCD such as nuclear condensation and DNA nicking. However, at 2 h, the proportion of cells in different phases of the cell cycle was comparable in both sets. This finding indicated that nuclear changes are switched on by alterations in the cell membrane and mitochondria and therefore can be considered as a relatively late phase of PCD.
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The treatment of promastigotes with racemoside A (10 µg ml–1) demonstrated a time-dependent increase in nuclear DNA fragmentation as shown by dUTP–FITC binding (Fig. 7). After 18 h of treatment, the degree of DNA nicking (MFI=9.05) was marginally increased compared with untreated promastigotes (MFI=6.91), which served as a control. However, after 24 h, the degree of DNA nicking was amplified (MFI=19.41) and the percentage of dUTP–FITC-positive cells increased to 23.07 % compared with 4.28 % of untreated cells (ethanol-fixed mammalian cell populations, supplied in the kit, served as positive and negative controls; data not shown).
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). This signal was orchestrated by a substantial depolarization (32.64 %) of the mitochondrial membrane after 6 h (Fig. 5), chromatin condensation (Fig. 3), cell-cycle arrest at the sub-G0/G1 phase after 24 h (Fig. 6) and TUNEL positivity after 24 h (Fig. 7).
In this study, we have shown for the first time that a newly identified, water-soluble, natural steroidal saponin, racemoside A (Fig. 1), had a leishmanicidal effect against promastigotes of both an SAG-sensitive and an SAG-unresponsive strain (Fig. 2a). This anti-leishmanial activity was almost seven times higher against intracellular amastigotes (Fig. 2b), with a high therapeutic ratio (Fig. 2c), making it a potent candidate for future chemotherapy.
Thus, we have conclusively demonstrated the leishmanicidal effect of racemoside A-induced PCD as shown by a number of biochemical and morphological techniques including annexin V and PI co-staining, changes in mitochondrial membrane potential, cell-cycle arrest and in situ DNA nicking. This study widens the scope for the design of new chemotherapeutic agents for better management of this disease.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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-hederin, ß-hederin and hederacolchiside A1, as compared to their action on mammalian cells cultured in vitro. Planta Med 66, 343–347.[CrossRef][Medline]This article has been cited by other articles:
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  H. Hillesland, A. Read, B. Subhadra, I. Hurwitz, R. McKelvey, K. Ghosh, P. Das, and R. Durvasula Identification of Aerobic Gut Bacteria from the Kala Azar Vector, Phlebotomus argentipes: A Platform for Potential Paratransgenic Manipulation of Sand Flies Am J Trop Med Hyg, December 1, 2008; 79(6): 881 - 886. [Abstract] [Full Text] [PDF]
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) and GE1F8R (
) and GE1F8R (
) were treated with amphotericin B (0–10 µg ml–1) as an experimental control and evaluated similarly. Each point corresponds to the mean±SD of at least three experiments in duplicate. (b) Evaluation of the anti-amastigote activity of racemoside A. Murine peritoneal macrophages were infected overnight with stationary-phase promastigotes (at a ratio of 1 : 5) of AG83 (
). After removal of non-phagocytosed parasites, infected macrophages were incubated with increasing concentrations of racemoside A (0–1 µg ml–1) for 72 h and the degree of parasite removal was analysed microscopically from Giemsa-stained slides as described in Methods. Leishmania-infected macrophages (
