New Insights into the Mechanism of Action of the Cyclopalladated Complex (CP2) in Leishmania: Calcium Dysregulation, Mitochondrial Dysfunction, and Cell Death

ABSTRACT

The current treatment of leishmaniasis is based on a few drugs that present several drawbacks, such as high toxicity, difficult administration route, and low efficacy. These disadvantages raise the necessity to develop novel antileishmanial compounds allied with a comprehensive understanding of their mechanisms of action. Here, we elucidate the probable mechanism of action of the antileishmanial binuclear cyclopalladated complex [Pd(dmba)(μ-N₃)]₂ (CP2) in Leishmania amazonensis. CP2 causes oxidative stress in the parasite, resulting in disruption of mitochondrial Ca²⁺ homeostasis, cell cycle arrest at the S-phase, increasing the reactive oxygen species (ROS) production and overexpression of stress-related and cell detoxification proteins, and collapsing the Leishmania mitochondrial membrane potential, and promotes apoptotic-like features in promastigotes, leading to necrosis, or directs programmed cell death (PCD)-committed cells toward necrotic-like destruction. Moreover, CP2 reduces the parasite load in both liver and spleen in Leishmania infantum-infected hamsters when treated for 15 days with 1.5 mg/kg body weight/day CP2, expanding its potential application in addition to the already known effectiveness on cutaneous leishmaniasis for the treatment of visceral leishmaniasis, showing the broad spectrum of action of this cyclopalladated complex. The data presented here bring new insights into the CP2 molecular mechanisms of action, assisting the promotion of its rational modification to improve both safety and efficacy.

INTRODUCTION

Leishmaniasis is a neglected parasitic disease caused by at least 20 species of the kinetoplastid genus Leishmania (1, 2) and is endemic in 98 countries (3). Cutaneous leishmaniasis (CL) is the most common clinical manifestation, while visceral leishmaniasis (VL) is a very severe systemic manifestation that can be fatal if left untreated (1, 2, 4). Causative CL species in the Old World are L. tropica, L. major, and L. aethiopica, in addition to L. infantum and L. donovani. In the Americas (New World), the species involved in CL are the L. mexicana species complex (especially L. mexicana, L. amazonensis, and L. venezuelensis) and the Viannia subgenus (most notably L. [Viannia] braziliensis, L. [V.] panamensis, L. [V.] guyanensis, and L. [V.] peruviana), in addition to L. major-like organisms and L. chagasi. For VL, the principal causative species of disease are the L. donovani species complex (i.e., L. donovani and L. infantum in the Old World, and L. chagasi in the New World) (5–7).

Available treatments for leishmaniasis have several limitations associated with high toxicity, difficult administration route, and low efficacy in areas of endemicity due to the emergence of resistant strains (4, 8–10). The adverse effects are mainly evident in leishmaniasis-HIV coinfection (1, 3). The current drugs for VL include pentavalent antimonials, amphotericin B (AmpB) and its lipid formulations (AmBisome), paromomycin, miltefosine, and drug combinations such as AmBisome/miltefosine, AmBisome/paromomycin, and miltefosine/paromomycin (2, 4, 11). For the CL, limited treatments are available (pentavalent antimonials, amphotericin B, and pentamidine) in comparison to VL, where these drugs are only recommended for the treatment of specific forms (2, 12). These challenges associated with just a few current pharmaceuticals highlight the urgent need to develop novel, safe, and effective leishmaniasis treatment drugs.

Thus, in order to contribute to new molecules that overcome the problems listed above, different strategies to identify new drugs against Leishmania spp. have been used (13–18). Many primary screenings of different compound libraries (natural products or synthetic molecules) have identified and validated hits (15, 19–32). Metal complexes with known antitumor bioactivity have been tested to treat various neglected diseases; some of them have exhibited antitrypanosomatid effects (21, 24–26, 28–30, 33–43). Moreover, the insertion of metal centers in antiparasitic drug structures is a strategy to increase their pharmacological activity by affecting multiple targets simultaneously (26). Au^III and Pd^II cyclometallated compounds and oxorhenium(V) complexes that inhibit different cysteine proteases of Trypanosoma cruzi and Leishmania spp. were developed as reported by Fricker and colleagues (25). Other authors reported the in vitro and in vivo leishmanicidal and trypanocidal activity of some Pd^II complexes (29, 30, 33, 35–38, 43–46). However, not all of these reports addressed the possible mechanism of action of these compounds. In general, studies involving Pd^II complexes reported that these compounds induce arrest of the cell cycle of parasites, generation of reactive oxygen species (ROS), interaction with DNA by electrostatic forces, irreversible inhibition of trypanothione reductase and cysteine protease, and inhibition of topoisomerase I (25, 30, 38, 43, 45, 46). The palladacycle compounds [Pd₂Cl₂(C²,N-dmpa)₂(μ-dppe)] (DPPE 1.1) and [Pd(C²,N-dmpa)(dppe)]Cl (DPPE 1.2), where dmpa is S(-)-N,N-dimethyl-1-phenethylamine and dppe is 1,2-bis(diphenylphosphino)ethane, reduced the parasite load of L. amazonensis-infected mice and reported cathepsin B and cysteine protease as their targets. However, the cell death mechanism induced by DPPE 1.1 and DPPE 1.2 has not been investigated (35–37). Other studies of DPPE 1.1 have described the cascade of effects produced after parasite treatment and showed that they also displayed action against other organisms (33, 34, 47). DPPE 1.1 exerts an apoptosis-like death in T. cruzi trypomastigotes and causes mitochondrion disruption (33); in Paracoccidioides lutzii and Paracoccidioides brasiliensis, the complex induced remarkable chromatin condensation, DNA degradation, superoxide anion production, metacaspase activity, and apoptosis- and autophagy-like mechanisms (34). In murine and cisplatin-resistant human tumor cells, DPPE 1.1 interacts with mitochondrial membrane thiol groups and induces the intrinsic apoptotic pathway (47).

Previously, we demonstrated that the binuclear cyclopalladated complex [Pd(dmba)(μ-N₃)]₂ (CP2) delivers in vivo leishmanicidal activity in L. amazonensis-infected mice as a CL model via inhibition of DNA topoisomerase 1B (30). In this study, we identified the downstream effects of the Leishmania topoisomerase IB inhibition by CP2 and its probable mechanism of action against L. amazonensis. Here, we observed that CP2 increased ROS and cytosolic Ca²⁺ levels and collapsed the mitochondrial membrane potential, leading the parasite to promote necrosis or leading programmed cell death (PCD)-committed cells toward necrotic-like destruction. In addition, CP2 causes an alteration in the levels of translation, stress-response proteins, and ROS detoxification in L. amazonensis. Finally, we demonstrated that CP2 not only has in vivo leishmanicidal activity on a CL model but also displayed effective leishmanicidal activity against L. infantum-infected hamsters, a VL model, which demonstrates the widespread potential of this cyclopalladated complex.

RESULTS

CP2 displayed in vitro and in vivo antileishmanial activity against L. infantum.

To evaluate the spectrum of action of CP2 beyond the effects previously reported (30), we analyzed the in vitro and in vivo efficacy of the compound using L. infantum as the causative agent of visceral leishmaniasis. CP2 displayed a high antileishmanial activity and selectivity index (SI) against both the insect promastigote stage (50% inhibitory concentration [IC₅₀], 4.0 μmol liter⁻¹; SI, 126.1) and the intracellular amastigote forms (IC₅₀, 4.7 μmol liter⁻¹; SI, 107.6) as reported in Table 1. The SI values were calculated using our previously reported values of cytotoxicity of CP2 against murine peritoneal macrophages (30).

TABLE 1.

Antileishmanial activity of CP2 against Leishmania infantum (IC₅₀)^a

Compound	L. infantum IC50 ± SD (SI)b
	Promastigote	Amastigote
CP2	4.0 ± 0.4 (126.1)	4.7 ± 0.1 (107.6)
AmpB	0.9 ± 0.1 (25.1)	2.9 ± 0.1 (7.7)

^aData are the mean and standard deviation from three independent experiments. The results are expressed in μmol liter⁻¹.

^bThe selectivity index (SI, indicated in parentheses) was calculated as the CC₅₀/IC₅₀ of CP2. P < 0.05 for all values.

L. infantum-infected golden hamsters were used as a VL model. The animals were treated for 15 days with CP2 (0.75 or 1.5 mg/kg body weight/day) or AmpB (20 mg/kg/day), and the parasite load of both spleen and liver was determined (Fig. 1). A dose of 1.5 mg/kg/day of CP2 caused an ∼50% reduction in the parasite load of both liver and spleen similar to AmpB (at a dose 13-fold higher than CP2), without any alteration in biochemical markers of liver and kidney function (see Fig. S1 at http://hdl.handle.net/11449/214617), following previous work (30).

FIG 1.

Parasite load of L. infantum-infected hamsters treated with CP2 or AmpB. Hamsters infected with L. infantum promastigotes in the stationary growth phase were treated with CP2 or AmpB commencing 75 days postinfection and lasting for 15 days. The parasite load was determined by the limiting dilution method at the end of the treatment in (a) the spleen and (b) the liver. The data are expressed as the mean ± standard deviation (SD). *, statistical significance of the difference relative to the untreated infected group (P < 0.05) was determined by ANOVA with Tukey’s post hoc test.

CP2 causes an alteration in the levels of stress-response proteins, protein translation, and ROS detoxification in Leishmania.

Two-dimensional SDS-PAGE comparative analysis was performed to identify differentially expressed proteins in the presence or absence of CP2. L. amazonensis promastigotes at the logarithm growth phase were treated with 13.3 μmol liter⁻¹ of CP2 for 72 h, a concentration corresponding to the previously determined IC₅₀ value (30). The protein extracts obtained were separated by two-dimensional SDS-PAGE at a pH ranging from 4 to 7 (Fig. S2 at http://hdl.handle.net/11449/214617).

A total of 52 differentially expressed proteins identified through mass spectrometry analyses (Table S1 at http://hdl.handle.net/11449/214617) are related to chaperons/protein folding (48%), mitochondrial respiratory chain (17%), and ROS detoxification (14%), in addition to α- and β-tubulins. In order to minimize possible artifacts, the spots were selected after data filtration based on two factors, a P value of less than 0.5 with Student’s t test comparing protein samples obtained in the presence or absence of CP2 and expression differences higher than 2-fold.

Among the overexpressed proteins, CP2 increased the levels of putative chaperons by at least 3-fold, including HSP70, LMXM_28_2770 (9-fold), two isoforms of putative calreticulin, LMXM_30_2600 (7-fold [pI 4.14] and 2-fold [pI 4.53]), and protein disulfide-isomerase (PDI), LMXM_36_6940 (3-fold). For the group of redox proteins, trypanothione reductase, LMXM_05_0350, and the peroxidoxin tryparedoxin peroxidase, LMXM_23_0040, increased by 6- and 4-fold, respectively.

Some proteins related to parasites’ mitochondria were found overexpressed in the presence of CP2, such as mitochondrial cytochrome c oxidase subunit IV, LMXM_12_0670, and ribonucleoprotein p18, LMXM_15_0275, while others, such as putative mitochondrial chaperone heat shock 70-related protein 1 (mHSP70-1), LMXM_29_2550, were not expressed in the cells treated with CP2.

In the functional category of protein synthesis, the putative translation elongation factor 1-beta, LMXM_33_0840, was 11-fold higher than that of to the control, while the elongation factor 1-alpha, LMXM_17_0080, the elongation factor 2, LMXM_36_0180, and the 60S acidic ribosomal protein P0, LMXM_27_1380, were only expressed in the absence of CP2. The putative carboxypeptidase, LMXM_32_2540, in the category of amino acid metabolism, was increased 2-fold. Additionally, reverse transcriptase quantitative PCR (RT-qPCR) data also show a correspondence between transcriptional and protein levels for calreticulin, PDI, and tryparedoxin peroxidase (Fig. S3 at http://hdl.handle.net/11449/214617) induced by CP2 (Fig. 2).

FIG 2.

Relative gene expression of calreticulin, PDI, and tryparedoxin peroxidase of Leishmania amazonensis promastigotes after 72 h of exposure to CP2. The relative gene expression determined by RT-qPCR was calculated using the 2^-–ΔΔCT method, using kDNA mRNA expression as a reference and the L. amazonensis (La) in the absence of CP2 as the calibrator (see the supplemental material at http://hdl.handle.net/11449/214617). The data were expressed as the average ± SD. *, statistical significance of the difference relative to La in the absence of CP2, control (P < 0.001), was determined by ANOVA with Tukey’s post hoc test. La-CP2 is the L. amazonensis treated with 13.3 μmol liter⁻¹ CP2.

CP2 arrests cell cycle progression at S-phase in Leishmania.

Previously, we demonstrated that CP2 inhibited the cleavage step of DNA topoisomerase 1B of Leishmania (30), leading to the accumulation of DNA damage and arrest in the cell cycle progression. To investigate the effect of CP2 on cell cycle progression, hydroxyurea (HU)-synchronized L. amazonensis promastigotes were treated with CP2 or CP2 plus the ROS inhibitor N-acetyl l-cysteine (NAC), and the progression of the cell cycle was followed by determining the parasite DNA content by flow cytometry (Fig. 3).

FIG 3.

CP2 effect on the cell cycle of Leishmania amazonensis. Promastigotes in the mid-log growth phase were synchronized by adding 5 mmol liter⁻¹ HU for 8 h and then transferred to an HU-free medium containing 0.03% DMSO (control). The parasites were treated with CP2 (4× IC₅₀, 53.2 μmol liter⁻¹), 20 μmol liter⁻¹ NAC or CP2 (4× IC₅₀, 53.2 μmol liter⁻¹ or 10× IC₅₀, 133 μmol liter⁻¹) plus 20 μmol liter⁻¹ NAC. Cells were collected at 0, 2, 4, 6, and 8 h, and the DNA content was measured by flow cytometry. Each histogram represents the data of 50,000 events; 2n and 4n indicate nonreplicated and replicated DNA, respectively. The percentage of cells in G₁, S, and G₂/M phases is indicated for cells before treatment with HU (pre-HU) and at 2 h and 8 h after the removal of HU. HU, hydroxyurea; NAC, N-acetyl-l-cysteine. P < 0.01.

The data obtained demonstrate that CP2 induced the accumulation of a higher proportion of cells in the S-phase, indicating an arrest of the cell cycle at this phase after CP2 treatment at 53.2 μmol liter⁻¹. Notably, this effect is not abolished by the addition of NAC. Moreover, promastigotes in the presence of 133 μmol liter⁻¹ CP2 (10× IC₅₀) increased the proportion of cells with 4n (see Fig. 3) DNA content, indicating that the effect of CP2 on the parasite cell cycle progression occurred independently of ROS.

CP2 increases ROS levels in Leishmania.

Promastigotes were exposed for 60 min to different concentrations of CP2 to determine its ability to increase cellular ROS levels every 5 min. CP2 increased ROS production in a dose-dependent manner (Fig. 4). This finding correlates with the proteomic data that showed overexpression of trypanothione reductase, peroxiredoxin, and tryparedoxin peroxidase, enzymes involved in the trypanothione-mediated hydroperoxide metabolism for detoxification of endogenous or exogenous oxidative agents (48).

FIG 4.

CP2-dependent ROS generation in L. amazonensis. Promastigote forms of L. amazonensis were treated with CP2 (0.5× IC₅₀, 6.7 μmol liter⁻¹; IC₅₀, 13.3 μmol liter⁻¹; 2× IC₅₀, 26.6 μmol liter⁻¹ and 4× IC₅₀, 53.2 μmol liter⁻¹) for 60 min. ROS generation was measured spectrofluorimetrically using the probe H₂DFCDA. The parasites were treated with 0.1% DMSO as a control. Data are the mean ± SD from three independent experiments. *, statistically significant difference relative to DMSO control (P < 0.001); α, statistically significant difference relative to parasites treated with 0.5× IC₅₀ of CP2 (P < 0.01). Statistical significance was determined by ANOVA with Student-Newman-Keuls multiple-comparison test.

CP2 disrupts Ca²⁺ homeostasis in Leishmania in a dose-dependent manner.

Considering that oxidative stress is often associated with a rise in intracellular Ca²⁺ concentration ([Ca²⁺]_i) (49–52), we determined the effect of CP2 on [Ca²⁺]_i in single-promastigote cells by live-cell fluorescence imaging using the ratiometric Ca²⁺ indicator Fura-2/AM (Fig. 5). After CP2 addition, increases in [Ca²⁺]_i were observed in the majority of cells after a delay of 5 to 10 min. The subsequent addition of the Ca²⁺ ionophore ionomycin increased [Ca²⁺]_i to the maximum, and this occurred with no delay. The presence of an ionomycin response and maintenance of the Fura-2 dye load of the cells was used to validate that the cells remained viable during the experiment. The [Ca²⁺]_i responses to CP2 varied in magnitude, with an increase to near-maximal [Ca²⁺]_i levels (relative to the ionomycin response) in some promastigotes (Fig. 5a). In contrast, others exhibited small peaks and only partial increases in [Ca²⁺]_i (Fig. 5b). When the responses were averaged from all parasites in the imaging field, the profile [Ca²⁺]_i showed a progressive increase during the incubation period (Fig. 5c). Additionally, the percentage of parasites that exhibited changes in [Ca²⁺]_i during the incubation period with CP2 increased in a dose-dependent manner (Fig. 5d). In the presence of 3.8 μmol liter⁻¹, an increase in [Ca²⁺]_i was detected in 20% of cells during the 25 to 30 min time course of the experiment, whereas in the presence of 7.5 μmol liter⁻¹ and 15 μmol liter⁻¹ responses were detected in 60% and 98% of cells, respectively. Following the large [Ca²⁺]_i increase during continuing incubation with high doses of CP2, some cells showed a loss of the total Fura-2 signal, which was taken as an indicator of cell death (Fig. S4 at http://hdl.handle.net/11449/214617). Thus, the increase in Ca²⁺ levels appears to presage cell death in response to CP2 (Fig. 5c).

FIG 5.

Effect of CP2 on the [Ca²⁺]_i of Leishmania mexicana promastigotes. The parasites were cultivated until the mid-log growth phase and then loaded with 5 μmol liter⁻¹ Fura-2/AM in a loading buffer containing 1.3 mmol liter⁻¹ of CaCl₂. Changes in [Ca²⁺]_i were measured with Fura-2 excitation at 340/380 nm and emission at >510 nm and are plotted as the 340/380 ratio (Fura-2 ratio). (a and b) Representative single-cell traces of large (a) and small (b) [Ca²⁺]_i changes in response to 7.5 μmol liter⁻¹ CP2 treatment of promastigotes are shown. Ionomycin (10 μmol liter⁻¹) was added at the end of each experiment to determine the maximal [Ca²⁺]_i response for each cell. The right panels shows pseudocolor images demonstrating the increase in fluorescence ratio related to an increase in [Ca²⁺]_i from blue (lowest ratio) to red (highest ratio) in the absence and presence of drugs. (c) Averaged trace for 100 cells in the imaging field. (d) Mean percentage of parasites with a change in [Ca²⁺]_i in the presence of 3.8, 7.5, or 15 μmol liter⁻¹ CP2; 0.1% DMSO was used as a control. Cell death counts reflect cells that showed total loss of Fura-2 by the end of the imaging experiment. Summary data are the mean ± SD from three independent experiments. α, statistically significant difference relative to dead cells in the presence of 15 μmol liter⁻¹ CP2; *, statistically significant difference between groups (P < 0.001) was determined by two-way ANOVA followed by Tukey’s multiple-comparison test.

Removal of extracellular Ca²⁺ did not affect the percentage of promastigotes of L. mexicana exposed to CP2, in which increases in [Ca²⁺]_i were observed (Fig. 6a and b). These data show that CP2-induced [Ca²⁺]_i responses are observed in the absence of extracellular Ca²⁺, indicating that the observed [Ca²⁺]_i increase is caused by Ca²⁺ release from an intracellular Ca²⁺ store, such as the endoplasmic reticulum (ER), acidic compartments, or mitochondria. Of note, an increase in dead cells after CP2 addition was also still observed in the absence of extracellular Ca²⁺ (Fig. 6c).

FIG 6.

Removal of extracellular Ca²⁺ does not perturb CP2-induced [Ca²⁺]_i responses in Leishmania mexicana. (a and b) A representative experiment of promastigotes cultivated until the mid-log growth phase and then loaded with Fura-2/AM in a loading buffer with (a) or without (b) 1.3 mmol liter⁻¹ of CaCl₂ and treated with 7.5 μmol liter⁻¹ CP2 followed by 10 μmol liter⁻¹ ionomycin. Traces averaged from 100 individual parasites. (c) Mean of the percentage of parasites with an increase in [Ca²⁺]_i and the percentage of dead cells in the presence or absence of extracellular Ca²⁺ after CP2 addition. Summary data are the mean ± SD from three independent experiments. *, statistically significant difference between groups (P < 0.001) was determined by two-way ANOVA followed by Tukey’s multiple-comparison test.

The metal chelator TPEN does not suppress the CP2-dependent [Ca²⁺]_i response.

Since CP2 is a complex of Pd²⁺, and heavy metals can affect Ca²⁺ channels and pumps, we tested the effect of TPEN (N,N,N′,N′-tetrakis [2-pyridylmethyl] ethylenediamine), a permeable cell and highly selective heavy metal-chelating agent with low affinity for Mg²⁺ and Ca²⁺ (53, 54). L. mexicana promastigotes were exposed to 10 μmol liter⁻¹ TPEN followed by CP2 addition (3.8 and 7.5 μmol liter⁻¹) (Fig. 7). It was observed that TPEN does not inhibit the ability of CP2 to mobilize intracellular Ca²⁺ (Fig. 7a and b). CP2 (7.5 μmol liter⁻¹)-induced [Ca²⁺]_i responses were observed in ∼60% of cells in the presence or absence of TPEN (Fig. 7c). Heavy metals have been shown to alter the fluorescent properties of Ca²⁺ indicator dyes (53). The addition of CP2 does not alter the fluorescence of Fura-2 in Leishmania, and TPEN does not perturb the Ca²⁺ response to this agent, which indicates that Pd²⁺ is coordinated with N,N-dimethylbenzylamine (dmba), and azide (N₃–) in the active species of CP2, which contributes to its effect on [Ca²⁺]_i mobilization. Notably, the dmba by itself did not exhibit leishmanicidal activity, as previously demonstrated (29). Nevertheless, it cannot be ruled out that CP2 can participate in competing ligand exchange reactions with other molecules or bridge splitting reactions during the experiments, affording new species, which may be responsible for the observed activity.

FIG 7.

Effect of CP2 on the L. mexicana [Ca²⁺]_i in the presence of TPEN, a heavy metal chelating agent. (a) Representative experiment of promastigotes treated with 3.8 or 7.5 μmol liter⁻¹ CP2 followed by 10 μmol liter⁻¹ ionomycin; (b) promastigotes treated with 10 μmol liter⁻¹ TPEN plus 3.8 or 7.5 μmol liter⁻¹ CP2, followed by 10 μmol liter⁻¹ ionomycin. Each trace is the average from at least 100 individual parasites. (c) Summary data of the percentage of cells with increased [Ca²⁺]_i; 0.1% DMSO was used as a control. Data are the mean ± SD from three independent experiments. *, statistically significant difference relative to DMSO control (P < 0.001) was determined by two-way ANOVA followed by Tukey’s multiple-comparison test.

CP2 induces Ca²⁺ release from mitochondria but not ER or acidic Ca²⁺ pools in Leishmania.

Fura-2/AM-loaded promastigotes in the presence of extracellular Ca²⁺ were exposed to nigericin (55) or cyclopiazonic acid (CPA) (56), followed by the addition of CP2 in order to determine the Ca²⁺ store targeted by CP2. The addition of nigericin, which releases Ca²⁺ from acidic compartments such as the acidocalcisome and acidic vesicles, resulted in an elevation of [Ca²⁺]_i in ∼60% of the parasites. Nevertheless, an increase in [Ca²⁺]_i was still observed after adding CP2 to nigericin-treated cells in 90% of the cells. Also, in cells, that responded to nigericin, an additional increase in response to CP2 was observed in ∼77% (Fig. 8a and b). Thus, CP2 does not act by mobilizing Ca²⁺ from acidic pools in Leishmania.

FIG 8.

Effects of CP2 on [Ca²⁺]_i in Leishmania mexicana after the addition of nigericin. (a) Representative experiment of promastigotes treated with 2.5 μmol liter⁻¹ nigericin, followed by 7.5 μmol liter⁻¹ CP2 and 10 μmol liter⁻¹ ionomycin. The trace shows an average of at least 100 individual parasites. (b) Mean percentage of parasites with a change in [Ca²⁺]_i in the presence of nigericin, subsequent CP2 addition, and those that responded to both nigericin and CP2. Data are the mean ± SD from three independent experiments. There are no statistically significant differences between groups determined by the one-way ANOVA multiple-comparison test (P < 0.05).

CPA, a specific inhibitor of the sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) (56), was used to investigate whether CP2 releases Ca²⁺ from the ER. Ca²⁺ responses in Fura-2-loaded parasites were observed after the addition of both CPA and CP2, suggesting that the release of Ca²⁺ caused by these two drugs is independent (Fig. 9a). Almost 60% of the parasites showed an increase in [Ca²⁺]_i in response to CPA, and 90% gave an [Ca²⁺]_i increase upon subsequent CP2 addition. The percentage of cells that responded to both CPA and CP2 was ∼70% (Fig. 9b).

FIG 9.

Effects of CP2 on [Ca²⁺]_i in Leishmania mexicana after the addition of cyclopiazonic acid (CPA). (a) Representative experiment of promastigotes treated with 10 μmol liter⁻¹ CPA, followed by 7.5 μmol liter⁻¹ CP2 and 10 μmol liter⁻¹ ionomycin. The trace shows an average of at least 100 individual parasites. (b) Mean of the percentage of parasites with increases in [Ca²⁺]_i in the presence of CPA, subsequent addition of CP2, and those that respond to both CPA and CP2. Data are the mean ± SD from three independent experiments. *, statistically significant differences relative to CP2 after CPA addition (P < 0.05) were determined by one-way ANOVA multiple-comparison test.

The results described above indicate that the CP2-dependent elevation of intracellular Ca²⁺ is not due to release from acidocalcisomes or the ER. To determine whether mitochondria might be the Ca²⁺ source, promastigotes were exposed to CP2 and the mitochondrial uncoupler FCCP (Fig. 10). The cell percentage with a [Ca²⁺]_i increase in response to CP2 was significantly decreased to about 10% by the pretreatment with FCCP (Fig. 10b). Similarly, prior treatment with CP2 decreased the response to subsequent FCCP addition. In both sequential addition paradigms, the percentage of parasites that responded to both FCCP and CP2 was only ∼5% (addition of FCCP followed by CP2 or CP2 followed by FCCP). Thus, these data suggest that CP2 mobilizes Ca²⁺ from a mitochondrial pool.

FIG 10.

Effect of CP2 on the Leishmania mexicana [Ca²⁺]_i before or after addition of FCCP (mitochondrial uncoupler). (a) Representative experiment of promastigotes treated with FCCP followed by CP2 (5 μmol liter⁻¹ and 7.5 μmol liter⁻¹, respectively) or CP2 followed by FCCP (7.5 μmol liter⁻¹ and 5 μmol liter⁻¹, respectively) and ionomycin (10 μmol liter⁻¹). Each trace is the average from at least 100 individual parasites. (b) Summary data showing the mean ± SD from three independent experiments. *, statistically significant difference relative to cells with [Ca²⁺]_i increase in the presence of CP2 (P < 0.05); α, statistically significant difference relative to cells with [Ca²⁺]_i increase in the presence of FCCP (P < 0.001) were determined by two-way ANOVA followed by Tukey’s multiple-comparison test.

CP2 depolarized the mitochondrial membrane potential of Leishmania.

To investigate whether CP2 alters the mitochondrial membrane potential (Δψ_m) of L. amazonensis, promastigotes were exposed to different concentrations of CP2 based on the IC₅₀ value for L. amazonensis previously determined (30) (IC₅₀, 13.3 μmol liter⁻¹ and 2× IC₅₀, 26.6 μmol liter⁻¹) for 60 min, and changes in Δψ_m were determined with JC-1. The spectrofluorometric data (Fig. 11) showed a decrease in the relative fluorescence intensity in all tested concentrations, indicating the membrane potential’s depolarization. Moreover, at a dose of 26.6 μmol liter⁻¹ of CP2, the decrease in the relative fluorescence intensity was similar to that caused by the mitochondrial uncoupler CCCP.

FIG 11.

Analysis of the mitochondrial membrane potential Δψ_m of L. amazonensis promastigotes. Promastigotes (2 × 10⁶/ml) were incubated with the potential-sensitive probe JC-1 (10 μmol liter⁻¹) for 10 min after exposure to different doses of CP2 (IC₅₀, 13.3 μmol liter⁻¹; 2× IC₅₀, 26.6 μmol liter⁻¹) for 60 min or CCCP (50 μmol liter⁻¹) 15 min before the addition of JC-1. For the untreated controls, promastigotes were incubated in the presence of 0.1% DMSO. Dose-dependent changes in relative Δψ_m values were expressed as the fluorescence ratio at 590 nm/530 nm. *, statistically significant difference relative to the control and between groups (P < 0.001). Statistical analysis was determined by ANOVA followed by Student-Newman-Keuls multiple-comparison test.

CP2 causes necrotic parasite death.

The cell death mechanism induced by CP2 was investigated using a dual acridine orange (AO)/ethidium bromide (EB) staining method (Fig. 12) (57). Cultures of L. amazonensis promastigotes were exposed to CP2 for 6, 24, or 48 h, 200 parasites of each sample were analyzed, and the percentages of viable cells (green), apoptotic-like cells (orange), or necrotic cells (red) were calculated (Fig. 12b to d). It was observed that 6 h post-CP2-treatment (Fig. 12b), promastigotes presented mainly apoptotic-like features (∼40%). However, exposure to CP2 for more extended periods (24 h and 48 h) produced ∼50% and 58% necrotic cells, respectively (Fig. 12c and d). Cell viability was significantly improved by NAC pretreatment, resulting in the prevention of apoptotic-like and necrotic cells (Fig. 12b to d). These data suggest that CP2-induced ROS formation triggers a programmed cell death response.

FIG 12.

CP2-induced necrotic cell death in L. amazonensis. Promastigotes were treated with 13.3 μmol liter⁻¹ CP2 for 6, 24, and 48 h in the presence and absence of 20 μmol liter⁻¹ NAC pretreatment for 2 h. The parasites were stained with acridine orange and ethidium bromide (AO/EB) and immobilized to be analyzed under a fluorescence microscope (Axio Imager A2; Zeiss), at ×100 magnification. (a) Staining of necrotic/apoptotic-like L. amazonensis promastigotes with AO/EB, where viable cells are green, necrotic cells red, and apoptotic-like cells yellow-orange. The images were captured with an Axio Cam MRm camera and processed using AxioVision software. (b to d) A total of 200 parasites from each sample were evaluated to determine the number of necrotic and apoptotic-like cells after (b) 6 h, (c) 24 h, and (d) 48 h of treatment. The control was 0.1% DMSO. Scale, 10 μm. *, statistically significant differences relative to the control and between treatments (P < 0.001) were determined by ANOVA with Student-Newman-Keuls multiple-comparison test.

DISCUSSION

We previously reported that the cyclopalladated complex CP2 inhibits Leishmania topoisomerase IB (30), which might lead to DNA damage, triggering cell cycle arrest and DNA repair (58–60). Here, we demonstrated that although CP2 is a ROS-inducing compound, the observed cell cycle arrest in S-phase, but not cell death, is ROS-independent, unlike DNA damage effects in Trypanosoma brucei, which are ROS inducible (61). Moreover, cells that have sustained DNA damage activate repair proteins, which cause NAD depletion and glycolysis inhibition. As a result, cells that become quickly depleted of ATP suffer necrotic cell death (62). Thus, we hypothesized that CP2 is causing several and simultaneous deleterious effects on the parasite.

Some cyclopalladated compounds act by targeting mitochondria (33, 34, 47, 63). This kind of compound could interact with thiol groups of mitochondrial membrane proteins, causing dissipation of the mitochondrial membrane potential, uncoupling of oxidative phosphorylation, increasing cytosolic Ca²⁺ and decreasing ATP levels, which lead to apoptosis, as reported by Serrano and colleagues (47). In this study, we demonstrated that CP2 increased ROS and cytosolic Ca²⁺ levels in Leishmania.

The trypanosomatid mitochondrion is a major source of ROS (64), and in order to deal with oxidative stress, the parasite produces several antioxidant molecules, including glutathione/trypanothione (64–66). Our proteomic analyses revealed other important overexpressed components, such as trypanothione reductase and tryparedoxin peroxidase induced by CP2. The trypanothione system is unique in trypanosomatids and protects them from oxidative damage and toxic heavy metals (67, 68). The combined action of trypanothione reductase, tryparedoxin, and tryparedoxin peroxidase is central to the maintenance of hydroperoxide metabolism (69). The trypanothione system is related to the mode of action and resistance to drugs containing metals, such as antimonials, by decreasing its thiol buffering capacity in Leishmania (68). In addition, elevated ROS levels are associated with an increase in peroxiredoxins involved in peroxynitrite protection (70). Thus, high peroxiredoxin levels observed in Leishmania after CP2 exposure might be related to the cell’s attempt to stabilize the mitochondrial membrane potential (71), wherein mitochondrial dysfunction and mitochondrial permeability transition induction are candidate intermediate steps in cell death (71, 72).

Oxidative membrane alterations can result in a secondary intracellular Ca²⁺ increase responsible for the irreversible disruption of membrane continuity (72). Calcium homeostasis is crucial for the correct functioning of mitochondria, and many antileishmanial agents exert their cytotoxic effects through the disruption of Ca²⁺ homeostasis in the parasite (49, 64). Ca²⁺ is stored in the acidocalcisomes, the ER, and the mitochondrion in trypanosomatids, keeping the cytosolic Ca²⁺ constant. Disruption of cytosolic Ca²⁺ signaling (73) and mitochondrial Ca²⁺ (73) may cause the parasite’s death or interfere with its virulence. Ca²⁺ transport mechanisms common to eukaryotic cells operate in the mitochondria of trypanosomatid parasites, such as the mitochondrial calcium uniporter (MCU), the voltage-dependent anion channel (VDAC1), and the Ca²⁺/H⁺ exchanger (CHX) (73, 74). VDAC1 in the outer mitochondrial membrane permits the Ca²⁺ influx into the intermembrane space and MCU in the inner mitochondrial membrane transport Ca²⁺ ions into the inner mitochondrial space (75). Efflux of Ca²⁺ to the cytosol from the inner mitochondrial space occurs via the Ca²⁺/H⁺ exchanger. The transport of Ca²⁺ into the mitochondria is membrane potential-dependent, and compounds that perturb the mitochondrial membrane potential result in Ca²⁺ release from the mitochondria into the cytosol as observed for FCCP. We demonstrated that promastigotes treated with CP2 suffer a loss of mitochondrial membrane potential and release of mitochondrial Ca²⁺ into the cytosol. Our results demonstrated that the high levels of [Ca²⁺]_i observed in Leishmania exposed to CP2 were derived from the mitochondrion as a result of the loss of mitochondrial membrane potential.

Increased Leishmania calreticulin and PDI levels after CP2 exposure were also observed. Calreticulin is a protein that presents high affinity for Ca²⁺, contributing to the maintenance of Ca²⁺ homeostasis. However, its Ca²⁺ affinity decreases in the presence of PDI (76), leading to a rapid disturbance in its homeostasis, which can be translated into cell death processes (77, 78).

The cyclopalladated compound DPPE 1.1, and not Pd²⁺, induced mitochondrial permeabilization and elevation in the cytosolic Ca²⁺ levels from intracellular pools, since PdCl₂ was not able to promote the same effect on the cation permeabilization (47). Similarly, the heavy metal ion chelator TPEN used here could not prevent the CP2-induced cytosolic Ca²⁺ increase, suggesting that the observed effect was not due to free Pd²⁺. Additionally, the [Pd(C²,N-dmba)(N₃)] CP2’s moiety is important for the Ca²⁺ to unbalance, since free dmba did not exert an antileishmanial effect (29).

Protein folding is sensitive to changes in Ca²⁺ flux and the parasite’s exposure to reducing agents (79). Proteins such as HSP70, HSP10, GRP78, and PDI play an important role in repairing the correct three-dimensional structure of unfolded proteins or forming aggregates due to stress. The observed increment of these proteins in CP2-treated parasites suggests the parasite’s efforts to refold proteins and diminish the harmful effect of CP2 to the cell. Moreover, GRP78, HSP70, and PDI are dependent on ATPase activity (80) and are strongly related to the increase in proteins secreted inside ER and degraded by the proteasome. The above-mentioned findings will be the subject of analysis in future studies.

In trypanosomatids, apoptosis-like cell death mechanisms associated with a lack of molecular markers and conditions are not fully understood (81). It is known that low ROS levels promote apoptosis events, while necrosis is observed at high ROS levels (72, 82–84). Our data showed that Leishmania exposed to CP2 increased ROS levels for 60 min, which favored the necrotic process as seen in parasites exposed to CP2 for 24 h. Moreover, the percentage of living or apoptotic-like promastigotes after CP2 exposure increased in the presence of NAC. Indeed, our previous work (30) reported an inflammatory process in a L. amazonensis-infected BALB/c mice treated with CP2, which might corroborate the high number of necrotic cells detected after parasite exposure to CP2 for 48 h.

It is important to consider that Ca²⁺ also plays an important role in cell death mechanisms inducing both apoptosis (low Ca²⁺ levels) or necrosis (high Ca²⁺ levels) (72, 82, 84). We have shown here that promastigotes exposed to CP2 displayed changes in [Ca²⁺]_i, followed by cell death. Thus, high ROS and [Ca²⁺]_i levels were observed ∼10 min after CP2 addition, which suggested that ROS production is shortly followed by release of mitochondrial Ca²⁺. These data suggest that ROS might be playing an essential role in the signaling/execution of the observed cell death mechanism and that Ca²⁺ acts on the observed mitochondrial changes, such as membrane depolarization, pointing out the parasite mitochondrion as central in orchestrating cell fate after triggering cell death signaling induced by CP2 (34, 49, 85).

Finally, beyond the previously reported antileishmanial efficacy of CP2 in a CL mouse model (30), here, we demonstrated that CP2 is also able to diminish the parasite load in a hamster model, as a visceral leishmaniasis model, indicating the broad potential of CP2 to exert its antileishmanial activity targeting other leishmaniasis clinical manifestations. Thus, the work presented here will help to promote rational drug modifications and thus contribute to the pipeline of leishmaniasis drug discovery.

MATERIALS AND METHODS

Compounds.

The binuclear cyclopalladated complex [Pd(C²,N-dmba)(μ-N₃)]₂ (dmba, N,N-dimethylbenzylamine), here called CP2, was obtained as previously described (86). Stock solutions of CP2, amphotericin B (AmpB; Cristalia, São Paulo, Brazil), nigericin (Sigma-Aldrich), FCCP (Sigma-Aldrich), ionomycin (Sigma-Aldrich), CPA (Calbiochem, San Diego, CA), and TPEN (Sigma-Aldrich) were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) and further diluted in culture medium (final 0.1% DMSO). Stock solutions were kept at −20°C.

Biological assay.

(i) Parasite culture. Promastigotes of L. mexicana strain MNYC/BZ/62/M379 and L. infantum strain MHOM/BR/1972/LD were maintained in Schneider’s insect medium (Sigma-Aldrich), and L. amazonensis strain MPRO/BR/1972/M1841-LV-79 was maintained in liver-infusion tryptose (LIT) medium (87) at 28°C, supplemented with 10% heat-inactivated bovine serum (iFBS; Gibco/Invitrogen) and 1% penicillin/streptomycin (Sigma-Aldrich).

(ii) Animals.

Male Swiss albino mice used to evaluate in vitro leishmanicidal activity against intracellular amastigotes were obtained from São Paulo State University (UNESP, Araraquara, São Paulo, Brazil). Male golden hamsters (Mesocricetus auratus) used in in vivo assays were acquired from ANILAB (Animais de Laboratório Criação e Comércio Ltda., Paulínia, São Paulo, Brazil). All animals were maintained in single-sex cages under a 12-h light/12-h dark cycle in a controlled-temperature room (22 ± 2°C), and they were fed ad libitum. The Ethics Committee for Animal Experimentation of São Paulo State University (UNESP), School of Pharmaceutical Sciences (CEUA/FCF/CAr, 18/2015 and 44/2015) approved this study in agreement with the guidelines of Sociedade Brasileira de Ciência de Animais de Laboratório (SBCAL) and Conselho Nacional de Controle da Experimentação Animal (CONCEA).

Antileishmanial in vivo assay.

To analyze the potential spectrum of action of CP2, we evaluated in vitro and in vivo leishmanicidal activity of the cyclopalladated complex against L. infantum. The MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay evaluated the susceptibility of L. infantum promastigotes, and evaluation of intracellular amastigotes forms was done according to a previously reported methodology (29, 30), followed by evaluation of in vivo leishmanicidal activity of CP2 against L. infantum-infected hamsters according to a previously established methodology (88). Briefly, 8-week-old male golden hamsters were intraperitoneally infected with 2 × 10⁸ promastigotes of L. infantum in the stationary phase of growth and randomly separated into six groups containing five animals per cage. Then, 75 days postinfection, the animals received 15 daily intraperitoneal doses of CP2 (1.5 or 0.75 mg/kg/day), the reference drug AmpB (20 mg/kg/day), or phosphate-buffered saline (PBS) (vehicle); untreated infected animals and noninfected animals were also evaluated. The animals were euthanized at the end of treatment. According to a previously established methodology, the blood was collected by cardiac puncture to obtain blood serum samples to analyze liver and renal function biomarkers (88). Parasite burden in the spleen and liver was determined by the limiting dilution methodology as previously described (88, 89).

Proteomic analyses.

For comparative proteomic analysis, 1 × 10⁷ parasites ml⁻¹ of promastigote forms of L. amazonensis in the mid-log phase were treated with 13.3 μmol liter⁻¹ CP2 (IC₅₀ of CP2 previously reported) (30) for 72 h. The extraction of total proteins of promastigotes was carried out in the presence (treated cells) and absence of CP2 (untreated cells). The parasite cultures were made in biological triplicates.

Protein extraction.

Promastigote forms of L. amazonensis untreated and treated with CP2 were centrifuged at 2,000 × g for 10 min at 4°C. The pellet was washed three times with tryps wash (100 mmol liter⁻¹ NaCl, 3 mmol liter⁻¹ MgCl₂, and 20 mmol liter⁻¹ Tris-HCl, pH 7.5) and resuspended in 500 μl of lysis solution (7 mol liter⁻¹ urea, 2 mol liter⁻¹ thiourea, 4% CHAPS, 2% IPG buffer 3-10, and 40 mmol liter⁻¹ DTT) (90) containing a protease inhibitor cocktail (cOmplete, mini-protease inhibitor cocktail; Roche) for 1 h under constant stirring at 4°C. Finally, the supernatant was collected by centrifugation at 14,000 × g for 3 min at 4°C and stored at −80°C until the time of use. Protein extract quantification was performed with the 2D Quant kit (GE Healthcare) using the Infinite 200 PRO plate reader (Tecan).

Two-dimensional SDS-PAGE.

Two-dimensional (2D) gels were run in triplicate according to standard procedures. pH 4 to 7 IPG buffer (Sigma-Aldrich) and bromophenol blue were added to 61.5 μg of protein and run on 24- cm pH 4 to 7 strips (GE Healthcare). The strips were equilibrated in equilibration buffer (6 mol liter⁻¹ urea, 75 mmol liter⁻¹ Tris-HCl, pH 8.8, 29.3% glycerol, 2% SDS, and 0.002% bromophenol blue) containing 25 mg ml⁻¹ dithiothreitol (DTT) for 15 min under gentle stirring. The previous solution was discarded, and a new equilibration buffer containing 10 mg ml⁻¹ iodoacetamide was added for 15 min. For the first dimension, rehydrated strips containing protein extracts of L. amazonensis promastigotes were transferred to ceramic support (Ettan IPGphor Manifold; GE Healthcare) and subjected to 50 μA per strip for protein migration using the Ettan IPGphor II isoelectric focusing system (GE Healthcare). The second dimension was run on 12.5% polyacrylamide gels (40 mA per gel) under continuous cooling (10°C) using an SE 600 Ruby and Multitemp IV instrument (GE Healthcare). The gels were stained with 0.1% Phast gel blue R solution (GE Healthcare) before scanning and analysis using ImageMaster 2D Platinum 7.0 software (GE Healthcare). Spots were cut, destained, and dried before trypsinization. Briefly, the spots were washed three times using 0.1 mol liter⁻¹ NH₄HCO₃ in 50% acetonitrile (ACN), followed by the addition of pure ACN for 15 min. Finally, the spots were air-dried and trypsinized with Trypsin Gold mass spectrometry grade (Promega) according to the manufacturer’s instructions.

Mass spectrometry (MS) analysis.

The analysis of tryptic peptides was performed with a nanoACQUITY UPLC device (Waters, Milliford, MA, USA) coupled to the mass spectrometer Xevo Q-TOF G2 (Waters). For this, the UPLC nanoACQUITY system was equipped with a column of high-strength silica (HSS) T3 (Acquity UPLC HSS T3 column 75 mm by 150 mm; 1.8 μm; Waters), previously equilibrated with 7% of the mobile phase B (100% ACN + 0.1% formic acid). The peptides were separated by a linear gradient of 7% to 85% of mobile phase B for 20 min with 0.35 μl min⁻¹ of flow at 45°C. The MS was operated in positive ion mode, with a data acquisition time of 20 min. The data obtained were processed using ProteinLynx GlobalServer 3.0 software (PLGS) (Waters). Protein identification of L. amazonensis was performed using the ion count algorithm integrated with the software and searched against the Leishmania mexicana UniProt (Universal Protein Resource, http://www.uniprot.org/proteomes/?query=Leishmania&sort=score, proteome ID UP000007259) protein database (analysis date, March 2016), used for comparison purposes since L. amazonensis and L. mexicana are phylogenetically related (6, 7, 91).

Cell cycle analysis.

Promastigotes of L. amazonensis were grown in an LIT medium at 28°C. Parasites in the exponential phase of growth (3 × 10⁶ parasites ml⁻¹) were incubated with 5 mmol liter⁻¹ hydroxyurea (HU) for 8 h and then transferred to HU-free medium supplemented with 0.03% DMSO, 20 μmol liter⁻¹ NAC (N-acetyl-l-cysteine; Sigma), 53.2 or 133 μmol liter⁻¹ CP2 (4× IC₅₀ or 10× IC₅₀), and 20 μmol liter⁻¹ NAC + 53.2 or 133 μmol liter⁻¹ CP2. Cells were collected after 2, 4, 6, or 8 h of treatment, washed with 1× PBS, and fixed in 30% PBS/70% methanol overnight at 4°C. Fixed cells were washed with 1× PBS and stained with PBS solution containing propidium iodide (10 μg ml⁻¹) and RNase A (10 μg ml⁻¹) at 37°C for 30 min. Flow cytometry data were collected using the BD FACSCanto 1 flow cytometer. Data were analyzed using FlowJo software.

Intracellular ROS measurement.

Intracellular ROS levels were compared after L. amazonensis exposure to CP2 based on the methodology previously described with some modifications (92). Briefly, parasites cultivated in Schneider’s insect medium until the mid-log growth phase were harvested, washed, and resuspended in modified 1 Hanks’ Balanced Salts (HBSS; Sigma) medium containing 1.3 mmol liter⁻¹ CaCl₂. Then, 1 × 10⁷ promastigotes ml⁻¹ were incubated in the dark with 20 mmol liter⁻¹ H₂DCFDA (2′,7′-dichlorodihydrofluorescein diacetate; Sigma) for 30 min, followed by treatment with different concentrations of CP2 based on the previously determined IC₅₀ value as follows (30): 6.7 μmol liter⁻¹ (0.5× IC₅₀), 13.3 μmol liter⁻¹ (1× IC₅₀), 26.6 μmol liter⁻¹ (2× IC₅₀), and 53.2 μmol liter⁻¹ (4× IC₅₀). A volume of 200 μl of parasites was transferred to the wells of black-bottom plates, and the fluorescence was measured at 530 nm (emission) and 480 nm (excitation) for 60 min (Infinite 200; Tecan), and the basal fluorescence was subtracted at the end of the process.

Live-cell Fura-2 Ca²⁺ imaging.

Promastigotes of L. mexicana in the mid-log phase (1 × 10⁷ parasites ml⁻¹) were harvested and subsequently washed in loading buffer (8.5 mmol liter⁻¹ Na₂HPO₄, 1.5 mmol liter⁻¹ KH₂PO₄, 137 mmol liter⁻¹ NaCl, 4 mmol liter⁻¹ KCl, 10 mmol liter⁻¹ HEPES 0.8 mmol liter⁻¹ MgCl₂, 0.8 mmol liter⁻¹ MgSO₄, 5 mmol liter⁻¹ glucose, and 2.4 mmol liter⁻¹ probenecid, pH 7.4), with and without 1.3 mmol liter⁻¹ CaCl₂, which contained 5 μmol liter⁻¹ Fura-2/AM (2-{6-[bis(carboxymethyl)amino]-5-(2-{2-[bis(carboxymethyl)amino]-5-methylphenoxy}ethoxy)-1-benzofuran-2-yl}-1,3-oxazole-5-carboxylic acid; Molecular Probes, Eugene, OR) and 2 μmol liter⁻¹ pluronic acid F-127 (Molecular Probes). The suspensions were incubated for 2 h at 28°C in the dark. Parasites were washed twice with the loading buffer to remove the extracellular dye and immobilized on coverslips previously treated with 2 μl Cell Tak (Cell-Tak cell and tissue adhesive; Corning) for 5 min. The coverslip containing the immobilized cells was transferred to the microscope stage (thermostatically regulated microscope chamber, open perfusion microincubator [PDMI-2]) at 28°C. The fluorescence of promastigote forms loaded with Fura-2/AM was captured at ×40 magnification, using an inverted microscope (Eclipse TE300; Nikon, Melville, NY) coupled to a digital camera (Hamamatsu EM-CCD ImagEM). Image acquisition was performed every 3 s at 340 nm and 380 nm excitation and 510 nm emission. A series of images generated in-frame was transformed into a video with the software NIS-Elements AR 4.20.02. The results were obtained from at least three independent experiments of 100 promastigote forms per assay. Responses to the addition of different compounds were captured in real time, followed by 10 μmol liter⁻¹ ionomycin to determine maximal fluorescence.

Measurement of the mitochondrial transmembrane potential Δψ_m.

Changes in the Δψ_m were measured spectrofluorimetrically using the cationic lipophilic dye 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazole carbocyanide iodide (JC-1; Sigma-Aldrich). Briefly, 2 × 10⁶ promastigotes ml⁻¹ of L. amazonensis were cultured for 60 min in the presence or absence of CP2. The parasites were harvested, resuspended in HBSS containing 1.3 mmol liter⁻¹ CaCl₂, and incubated with JC-1 (10 μmol liter⁻¹) for 10 min at 28°C. For the positive control, 50 μmol liter⁻¹ of the mitochondrial uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was added to untreated control cells 15 min before addition of JC-1. After washing twice with HBSS, fluorescence was measured at 530 nm and 590 nm using a spectrofluorometer (Tecan) with an excitation wavelength of 485 nm. The 590 nm/530 nm ratio values were plotted as the relative Δψ_m (93).

Cell death analysis using acridine orange/ethidium bromide (AO/EB) staining.

Promastigotes of L. amazonensis in the mid-log phase (1 × 10⁷ parasites ml⁻¹) were incubated with 13.3 μmol liter⁻¹ CP2 for 6 h, 24 h, and 48 h. Likewise, parasites were incubated with 0.1% DMSO as a control. In addition, parasites were pretreated with 20 μmol liter⁻¹ NAC for 2 h before CP2 addition. After the incubation period, the parasites were centrifuged for 10 min at 2,000 × g, washed with PBS, and resuspended in 200 μl of PBS. Then, 20 μl of cell suspension was stained with a mixture of ethidium bromide and acridine orange (100 μg ml⁻¹) and immobilized on a glass coverslip previously treated with Cell Tak, as described above. The labeled parasites were visualized by fluorescence microscopy (Axioplan-Zeiss) with ×100 magnification and a fluorescein isothiocyanate (FITC) filter (460 to 490 nm band-pass excitation filter and 510 to 560 nm emission) according to a previously described methodology (57). For the percentage quantification of each event, 200 cells from each sample were counted.

Statistical analysis.

The biological assays’ data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey and Student-Newman-Keuls multiple-comparison tests (Graph Pad InStat software and GraphPad Prism software). Differences were considered significant when P was ≤0.05.