Mitochondrial damage contribute to epigallocatechin-3-gallate induced death in Leishmania amazonensis

Epigallocatechin-3-gallate (EGCG), the most abundant flavonoid in green tea, has been reported to have antiproliferative effects on Trypanosoma cruzi however, the mechanism of protozoan action of EGCG has not been studied. In the present study, we demonstrate the mechanism for the antileishmanial activity of EGCG against Leishmania amazonensis promastigotes. Incubation with EGCG significantly inhibited L. amazonensis promastigote proliferation in a time- and dose-dependent manner. The IC(50) for EGCG at 120h was 0.063mM. Ultrastructural alterations of the mitochondria were observed in promastigote treated with EGCG, being the organelle injury reinforced by the decrease in rhodamine 123 fluorescence. The effects of several drugs that interfere directly with mitochondrial physiology in parasites such as Leishmania have been described. The unique mitochondrial features of Leishmania make this organelle an ideal drug target while minimizing toxicity. These data suggest mitochondrial collapse as a part of the EGCG mechanism of action and demonstrate the leishmanicidal effect of EGCG.     

Reactive oxygen species induced by proteasome inhibition in neuronal cells mediate mitochondrial dysfunction and a caspase-independent cell death

While increasing evidence shows that proteasome inhibition triggers oxidative damage, mitochondrial dysfunction and death in neuronal cells, the regulatory relationship among these events is unclear. Using mouse neuronal cells we show that the cytotoxicity induced by mild (0.25 μM) and potent (5.0 μM) doses of the proteasome inhibitor, N-Benzyloxycarbonyl-Ile-Glu (O-t-butyl)-Ala-leucinal, (PSI) involved a dose-dependent increase in caspase activation, overproduction of reactive oxygen species (ROS) and a mitochondrial dysfunction manifested by the translocation of the proapoptotic protein, Bax, from the cytoplasm to the mitochondria, membrane depolarization and the release of cytochrome c and the apoptosis inducing factor (AIF) from mitochondria to the cytoplasm and nucleus, respectively. Whereas caspase or Bax inhibition failed to prevent mitochondrial membrane depolarization and neuronal cell death, pretreatments with the antioxidant N-acetyl-l-cysteine (NAC) or overexpression of the antiapoptotic protein Bcl-xL abrogated these events in cells exposed to mild levels of PSI. These findings implicated ROS as a mediator of PSI-induced cytotoxicity. However, depletions in glutathione and Bcl-xL with potent proteasome inhibition exacerbated this response whereupon survival required the cooperative protection of NAC with Bcl-xL overexpression. Collectively, ROS induced by proteasome inhibition mediates a mitochondrial dysfunction in neuronal cells that culminates in death through caspase- and Bax-independent mechanisms. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Reactive oxygen species and imbalance of calcium homeostasis contributes to curcumin induced programmed cell death in Leishmania donovani

Curcumin, a polyphenol compound, has been recognized as a promising anti-cancer drug. The purpose of the present study was to investigate the cytotoxicity of curcumin to Leishmania donovani, the causative agent for visceral leishmaniasis. Flow cytometric analysis revealed that curcumin induced cell cycle arrest at G2/M phase. Incubation of Leishmania promastigotes with curcumin caused exposure of phosphatidylserine to the outer leaflet of plasma membrane. This event is preceded by curcumin-induced formation of reactive oxygen species (ROS) and elevation of cytosolic calcium through the release of calcium ions from intracellular stores as well as by influx of extracellular calcium. Elevation of cytosolic calcium is responsible for depolarization of mitochondrial membrane potential (ΔΨm), release of Cytochrome c into the cytosol and concomitant nuclear alterations that included deoxynucleotidyltransferase-mediated dUTP end labeling (TUNEL) and DNA fragmentation. Taken together, these data indicate that curcumin has promising antileishmanial activity that is mediated by programmed cell death and, accordingly, merits further investigation as a therapeutic option for the treatment of leishmaniasis.     

Reactive oxygen species and the mitochondrial signaling pathway of cell death

Reactive oxygen species (ROS) are produced as a by-product of cellular metabolic pathways and function as a critical second messenger in a variety of intracellular signaling pathways. Thus, a defect or deficiency in the anti-oxidant defense system on the one hand and/or the excessive intracellular generation of ROS on the other renders a cell oxidatively stressed. As a consequence, direct or indirect involvement of ROS in numerous diseases has been documented. In most of these cases, the deleterious effect of ROS is a function of activation of intracellular cell-death circuitry. To that end, involvement of ROS at different phases of the apoptotic pathway, such as induction of mitochondrial permeability transition and release of mitochondrial death amplification factors, activation of intracellular caspases and DNA damage, has been clearly established. For instance, the ROS-induced alteration of constitutive mitochondrial proteins, such as the voltage-dependent anion channel (VDAC) and/or the adenine nucleotide translocase (ANT) can induce the pro-apoptotic mitochondrial membrane permabilization. Not only do these observations provide insight into the intricate mechanisms underlying a variety of disease states, but they also present novel opportunities for the design and development of more effective therapeutic strategies.     

Camptothecin induced mitochondrial dysfunction leading to programmed cell death in unicellular hemoflagellate Leishmania donovani

The parasites of the order kinetoplastidae including Leishmania spp. emerge from most ancient phylogenic branches of unicellular eukaryotic lineages. In their life cycle, topoisomerase I plays a significant role in carrying out vital cellular processes. Camptothecin (CPT), an inhibitor of DNA topoisomerase I, induces programmed cell death (PCD) both in the amastigotes and promastigotes form of L. donovani parasites. CPT-induced cellular dysfunction in L. donovani promastigotes is characterized by several cytoplasmic and nuclear features of apoptosis. CPT inhibits cellular respiration that results in mitochondrial hyperpolarization taking place by oligomycin-sensitive F0-F1 ATPase-like protein in leishmanial cells. During the early phase of activation, there is an increase in reactive oxygen species (ROS) inside cells, which causes subsequent elevation in the level of lipid peroxidation and decrease in reducing equivalents like GSH. Endogenous ROS formation and lipid peroxidation cause eventual loss of mitochondrial membrane potential. Furthermore, cytochrome c is released into the cytosol in a manner independent of involvement of CED3/CPP32 group of proteases and unlike mammalian cells it is insensitive to cyclosporin A. These events are followed by activation of both CED3/CPP32 and ICE group of proteases in PCD of Leishmania. Taken together, our study indicates that different biochemical events leading to apoptosis in leishmanial cells provide information that could be exploited to develop newer potential therapeutic targets.     

Reactive oxygen species and mitochondrial diseases

A variety of diseases have been associated with excessive reactive oxygen species (ROS), which are produced mostly in the mitochondria as byproducts of normal cell respiration. The interrelationship between ROS and mitochondria suggests shared pathogenic mechanisms in mitochondrial and ROS-related diseases. Defects in oxidative phosphorylation can increase ROS production, whereas ROS-mediated damage to biomolecules can have direct effects on the components of the electron transport system. Here, we review the molecular mechanisms of ROS production and damage, as well as the existing evidence of mitochondrial ROS involvement in human diseases.     

Reactive oxygen species contribute to sleep apnea-induced hypertension in rats

In clinical studies, sleep apnea is associated with hypertension, oxidative stress, and increased circulating endothelin-1 (ET-1). We previously developed a model of sleep apnea by exposing rats to eucapnic intermittent hypoxia (IH-C) during sleep, which increases both blood pressure and plasma levels of ET-1. Because similar protocols in mice increase tissue and plasma markers of oxidative stress, we hypothesized that IH-C generation of reactive oxygen species (ROS) contributes to the development of ET-1-dependent hypertension in IH-C rats. To test this, male Sprague-Dawley rats were instrumented with indwelling blood pressure telemeters and drank either plain water or water containing the superoxide dismutase mimetic, Tempol (4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl, 1 mM). Mean arterial pressure (MAP) and heart rate (HR) were recorded for 3 control days and 14 treatment days with rats exposed 7 h/day to IH-C or air/air cycling (Sham). On day 14, MAP in IH-C rats treated with Tempol (107 +/- 2.29 mmHg) was significantly lower than in untreated IH-C rats (118 +/- 9 mmHg, P < 0.05). Tempol did not affect blood pressure in sham-operated rats (Tempol = 101 +/- 3, water = 101 +/- 2 mmHg). Immunoreactive ET-1 was greater in plasma from IH-C rats compared with plasma from sham-operated rats but was not different from Sham in Tempol-treated IH-C rats. Small mesenteric arteries from IH-C rats but not Tempol-treated IH-C rats had increased superoxide levels as measured by ferric cytochrome c reduction, lucigenin signaling, and dihydroethidium fluorescence. The data show that IH-C increases ET-1 production and vascular ROS levels and that scavenging superoxide prevents both. Thus oxidative stress appears to contribute to increases in ET-1 production and elevated arterial pressure in this rat model of sleep apnea-induced hypertension.     

Amentoflavone isolated from Selaginella sellowii Hieron induces mitochondrial dysfunction in Leishmania amazonensis promastigotes

Leishmaniasis chemotherapy is a bottleneck in disease treatment. Although available, chemotherapy is limited, toxic, painful, and does not lead to parasite clearance, with parasite resistance also being reported. Therefore, new therapeutic options are being investigated, such as plant-derived anti-parasitic compounds. Amentoflavone is the most common biflavonoid in the Selaginella genus, and its antileishmanial activity has already been described on Leishmania amazonensis intracellular amastigotes but its direct action on the parasite is controversial. In this work we demonstrate that amentoflavone is active on L. amazonensis promastigotes (IC₅₀ = 28.5 ± 2.0 μM) and amastigotes. Transmission electron microscopy of amentoflavone-treated promastigotes showed myelin-like figures, autophagosomes as well as enlarged mitochondria. Treated parasites also presented multiple lipid droplets and altered basal body organization. Similarly, intracellular amastigotes presented swollen mitochondria, membrane fragments in the lumen of the flagellar pocket as well as autophagic vacuoles. Flow cytometric analysis after TMRE staining showed that amentoflavone strongly decreased mitochondrial membrane potential. In silico analysis shows that amentoflavone physic-chemical, drug-likeness and bioavailability characteristics suggest it might be suitable for oral administration. We concluded that amentoflavone presents a direct effect on L. amazonensis parasites, causing mitochondrial dysfunction and parasite killing. Therefore, all results point for the potential of amentoflavone as a promising candidate for conducting advanced studies for the development of drugs against leishmaniasis.     

Hydrogen peroxide production and mitochondrial dysfunction contribute to the fusaric acid-induced programmed cell death in tobacco cells

Fusaric acid (FA), a non-specific toxin produced mainly by Fusarium spp., can cause programmed cell death (PCD) in tobacco suspension cells. The mechanism underlying the FA-induced PCD was not well understood. In this study, we analyzed the roles of hydrogen peroxide (H₂O₂) and mitochondrial function in the FA-induced PCD. Tobacco suspension cells were treated with 100 μM FA and then analyzed for H₂O₂ accumulation and mitochondrial functions. Here we demonstrate that cells undergoing FA-induced PCD exhibited H₂O₂ production, lipid peroxidation, and a decrease of the catalase and ascorbate peroxidase activities. Pre-treatment of tobacco suspension cells with antioxidant ascorbic acid and NADPH oxidase inhibitor diphenyl iodonium significantly reduced the rate of FA-induced cell death as well as the caspase-3-like protease activity. Moreover, FA treatment of tobacco cells decreased the mitochondrial membrane potential and ATP content. Oligomycin and cyclosporine A, inhibitors of the mitochondrial ATP synthase and the mitochondrial permeability transition pore, respectively, could also reduce the rate of FA-induced cell death significantly. Taken together, the results presented in this paper demonstrate that H₂O₂ accumulation and mitochondrial dysfunction are the crucial events during the FA-induced PCD in tobacco suspension cells.     

Reactive oxygen species-mediated endoplasmic reticulum stress and mitochondrial dysfunction contribute to polydatin-induced apoptosis in human nasopharyngeal carcinoma CNE cells

Previous studies revealed that polydatin, a natural small compound, possessed protective effect against ischemia/reperfusion injury and inflammation. However, the action and molecular mechanism of its potent anti-cancer activity remain poorly understood. In the present study, polydatin significantly killed several human tumor cell lines in a dose- and time-dependent manner. The compound also dose-dependently caused mitochondrial apoptosis in human nasopharyngeal carcinoma CNE cells. In addition, polydatin triggered endoplasmic reticulum (ER) stress and down-regulated the phosphorylation of Akt in CNE cells, while knock-down of CCAAT/enhancer-binding protein homologous protein (CHOP) dramatically abrogated the inactivation of Akt and reversed the pro-apoptotic effect of polydatin. Furthermore, polydatin provoked the generation of reactive oxygen species in CNE cells, while the antioxidant N-acetyl cysteine almost completely blocked the activation of ER stress and apoptosis, suggesting polydatin-induced reactive oxygen species is an early event that triggers ER stress mitochondrial apoptotic pathways in CNE cells. Taken together, these findings strongly suggest that polydatin might be a promising anti-tumor drug and our data provide the molecular theoretical basis for clinical application of polydatin.     

The leishmanicidal flavonols quercetin and quercitrin target Leishmania (Leishmania) amazonensis arginase

Polyamine biosynthesis enzymes are promising drug targets for the treatment of leishmaniasis, Chagas' disease and African sleeping sickness. Arginase, which is a metallohydrolase, is the first enzyme involved in polyamine biosynthesis and converts arginine into ornithine and urea. Ornithine is used in the polyamine pathway that is essential for cell proliferation and ROS detoxification by trypanothione. The flavonols quercetin and quercitrin have been described as antitrypanosomal and antileishmanial compounds, and their ability to inhibit arginase was tested in this work. We characterized the inhibition of recombinant arginase from Leishmania (Leishmania) amazonensis by quercetin, quercitrin and isoquercitrin. The IC(50) values for quercetin, quercitrin and isoquercitrin were estimated to be 3.8, 10 and 4.3 μM, respectively. Quercetin is a mixed inhibitor, whereas quercitrin and isoquercitrin are uncompetitive inhibitors of L. (L.) amazonensis arginase. Quercetin interacts with the substrate l-arginine and the cofactor Mn(2+) at pH 9.6, whereas quercitrin and isoquercitrin do not interact with the enzyme's cofactor or substrate. Docking analysis of these flavonols suggests that the cathecol group of the three compounds interact with Asp129, which is involved in metal bridge formation for the cofactors Mn(A)(2+) and Mn(B)(2+) in the active site of arginase. These results help to elucidate the mechanism of action of leishmanicidal flavonols and offer new perspectives for drug design against Leishmania infection based on interactions between arginase and flavones.     

Cellular stress induced by resazurin leads to autophagy and cell death via production of reactive oxygen species and mitochondrial impairment

Mitochondrial bioenergetics and reactive oxygen species (ROS) often play important roles in cellular stress mechanisms. In this study we investigated how these factors are involved in the stress response triggered by resazurin (Alamar Blue) in cultured cancer cells. Resazurin is a redox reactive compound widely used as reporter agent in assays of cell biology (e.g. cell viability and metabolic activity) due to its colorimetric and fluorimetric properties. In order to investigate resazurin‐induced stress mechanisms we employed cells affording different metabolic and regulatory phenotypes. In HL‐60 and Jurkat leukemia cells resazurin caused mitochondrial disintegration, respiratory dysfunction, reduced proliferation, and cell death. These effects were preceded by a burst of ROS, especially in HL‐60 cells which were also more sensitive and contained autophagic vesicles. Studies in Rho⁰ cells (devoid of mitochondrial DNA) indicated that the stress response does not depend on the rates of mitochondrial respiration. The anti‐proliferative effect of resazurin was confirmed in native acute myelogenous leukemia (AML) blasts. In conclusion, the data suggest that resazurin triggers cellular ROS production and thereby initiates a stress response leading to mitochondrial dysfunction, reduced proliferation, autophagy, and cell degradation. The ability of cells to tolerate this type of stress may be important in toxicity and chemoresistance. J. Cell. Biochem. 111: 574–584, 2010. © 2010 Wiley‐Liss, Inc.     

Reactive oxygen species induced by shear stress mediate cell death in Bacillus subtilis

Exposure of Bacillus subtilis to a shear rate of 1,482/s leads to a rapid loss of cell viability after 10 h of growth. Biochemical and molecular evidences provided below strongly suggest that cell death under high shear results from an apoptosis-like process similar to that described in eukaryotes, with activation of a caspase-3-like protease (C(3)LP) followed by DNA fragmentation. Shear stress leads to an increase in specific intracellular reactive oxygen species (siROS), possibly through activation of NADH oxidase (NOX). The formation of siROS precedes the activation of C(3)LP and DNA fragmentation, thus establishing siROS as the molecular link between shear stress and apoptosis-like cell death. A model is proposed in which NOX is viewed as being strategically placed on the plasma membrane of B. subtilis that senses and converts a mechanical force arising from shear stress into a chemical signal leading to activation of C(3)LP, DNA fragmentation, and thus, apoptosis-like cell death.     

Reactive oxygen species contribute to the release of volatile organic compounds from Chlamydomonas reinhardtii during programmed cell death

Acetic acid at pH 5.0 can induce programmed cell death (PCD) in Chlamydomonas reinhardtii cells, and abundant volatile organic compounds (VOCs) were released during the process. In this study, the caspase‐3‐like activity was determined during the PCD, and it was increased significantly after 1 h. During the PCD, the dynamic release of VOCs from the cells was analyzed, and the emissions of total VOCs were raised markedly and reached the highest level at 2 h. Among the seven types of VOCs, such as alkanes, alkenes, terpenoids, alcohols, aldehydes, ketones and esters, three oxygenated compounds (aldehydes, ketones and esters) showed the most significant increase. O₂^‐· and H₂O₂ were rapidly accumulated to high levels in the cells at the beginning of the PCD, but their content was reduced during the process. The activities of antioxidant enzymes were reduced gradually and even disappeared completely, demonstrating that the reduction of reactive oxygen species (ROS) may not be scavenged by the antioxidant enzyme system. ROS have an intense oxidation and scavenging ability to volatile compounds, and the oxidation results in the production of oxygenated compounds. Therefore, the abundant production of oxygenated compounds indicated that ROS may play an important role in the dynamic release of VOCs from C. reinhardtii cells during PCD.     

Tomatidine promotes the inhibition of 24-alkylated sterol biosynthesis and mitochondrial dysfunction in Leishmania amazonensis promastigotes

SUMMARY Leishmaniasis is a set of clinically distinct infectious diseases caused by Leishmania, a genus of flagellated protozoan parasites, that affects ～12 million people worldwide, with ～2 million new infections annually. Plants are known to produce substances to defend themselves against pathogens and predators. In the genus Lycopersicon, which includes the tomato, L. esculentum, the main antimicrobial compound is the steroidal glycoalkaloid α-tomatine. The loss of the saccharide side-chain of tomatine yields the aglycone tomatidine. In the present study, we investigated the effects of tomatidine on the growth, mitochondrial membrane potential, sterol metabolism, and ultrastructure of Leishmania amazonensis promastigotes. Tomatidine (0·1 to 5 μM) inhibited parasite growth in a dose-dependent manner (IC50=124±59 nM). Transmission electron microscopy revealed lesions in the mitochondrial ultrastructure and the presence of large vacuoles and lipid storage bodies in the cytoplasm. These structural changes in the mitochondria were accompanied by an effective loss of mitochondrial membrane potential and a decrease in ATP levels. An analysis of the neutral lipid content revealed a large depletion of endogenous 24-alkylated sterols such as 24-methylene-cholesta-5, 7-dien-3β-ol (5-dehydroepisterol), with a concomitant accumulation of cholesta-8, 24-dien-3β-ol (zymosterol), which implied a perturbation in the cellular lipid content. These results are consistent with an inhibition of 24-sterol methyltransferase, an important enzyme responsible for the methylation of sterols at the 24 position, which is an essential step in the production of ergosterol and other 24-methyl sterols.     

Reactive oxygen species production by forward and reverse electron fluxes in the mitochondrial respiratory chain

Reactive oxygen species (ROS) produced in the mitochondrial respiratory chain (RC) are primary signals that modulate cellular adaptation to environment, and are also destructive factors that damage cells under the conditions of hypoxia/reoxygenation relevant for various systemic diseases or transplantation. The important role of ROS in cell survival requires detailed investigation of mechanism and determinants of ROS production. To perform such an investigation we extended our rule-based model of complex III in order to account for electron transport in the whole RC coupled to proton translocation, transmembrane electrochemical potential generation, TCA cycle reactions, and substrate transport to mitochondria. It fits respiratory electron fluxes measured in rat brain mitochondria fueled by succinate or pyruvate and malate, and the dynamics of NAD(+) reduction by reverse electron transport from succinate through complex I. The fitting of measured characteristics gave an insight into the mechanism of underlying processes governing the formation of free radicals that can transfer an unpaired electron to oxygen-producing superoxide and thus can initiate the generation of ROS. Our analysis revealed an association of ROS production with levels of specific radicals of individual electron transporters and their combinations in species of complexes I and III. It was found that the phenomenon of bistability, revealed previously as a property of complex III, remains valid for the whole RC. The conditions for switching to a state with a high content of free radicals in complex III were predicted based on theoretical analysis and were confirmed experimentally. These findings provide a new insight into the mechanisms of ROS production in RC.     

Methyl jasmonate induces production of reactive oxygen species and alterations in mitochondrial dynamics that precede photosynthetic dysfunction and subsequent cell death

Methyl jasmonate (MeJa) is a well-known plant stress hormone. Upon exposure to stress, MeJa is produced and causes activation of programmed cell death (PCD) and defense mechanisms in plants. However, the early events and the signaling mechanisms of MeJa-induced cell death have yet to be fully elucidated. To obtain some insights into the early events of this cell death process, we investigated mitochondrial dynamics, chloroplast morphology and function, production and localization of reactive oxygen species (ROS) at the single-cell level as well as photosynthetic capacity at the whole-seedling level under MeJa stimulation. Our results demonstrated that MeJa induction of ROS production, which first occurred in mitochondria after 1 h of MeJa treatment and subsequently in chloroplasts by 3 h of treatment, caused a series of alterations in mitochondrial dynamics including the cessation of mitochondrial movement, the loss of mitochondrial transmembrane potential (MPT), and the morphological transition and aberrant distribution of mitochondria. Thereafter, photochemical efficiency dramatically declined before obvious distortion in chloroplast morphology, which is prior to MeJa-induced cell death in protoplasts or intact seedlings. Moreover, treatment of protoplasts with ascorbic acid or catalase prevented ROS production, organelle change, photosynthetic dysfunction and subsequent cell death. The permeability transition pore inhibitor cyclosporin A gave significant protection against MPT loss, mitochondrial swelling and subsequent cell death. These results suggested that MeJa induces ROS production and alterations of mitochondrial dynamics as well as subsequent photosynthetic collapse, which occur upstream of cell death and are necessary components of the cell death process.     

Reactive oxygen species and mitochondrial sensitivity to oxidative stress determine induction of cancer cell death by p21

p21(Waf1/Cip1/Sdi1) is a cyclin-dependent kinase inhibitor that mediates cell cycle arrest. Prolonged p21 up-regulation induces a senescent phenotype in normal and cancer cells, accompanied by an increase in intracellular reactive oxygen species (ROS). However, it has been shown recently that p21 expression can also lead to cell death in certain models. The mechanisms involved in this process are not fully understood. Here, we describe an induction of apoptosis by p21 in sarcoma cell lines that is p53-independent and can be ameliorated with antioxidants. Similar levels of p21 and ROS caused senescence in the absence of significant death in other cancer cell lines, suggesting a cell-specific response. We also found that cells undergoing p21-dependent cell death had higher sensitivity to oxidants and a specific pattern of mitochondrial polarization changes. Consistent with this, apoptosis could be blocked with targeted expression of catalase in the mitochondria of these cells. We propose that the balance between cancer cell death and arrest after p21 up-regulation depends on the specific effects of p21-induced ROS on the mitochondria. This suggests that selective up-regulation of p21 in cancer cells could be a successful therapeutic intervention for sarcomas and tumors with lower resistance to mitochondrial oxidative damage, regardless of p53 status.     

Miltefosine induces programmed cell death in Leishmania amazonensis promastigotes

In the current study, we evaluated the mechanism of action of miltefosine, which is the first effective and safe oral treatment for visceral leishmaniasis, in Leishmania amazonensis promastigotes. Miltefosine induced a process of programmed cell death, which was determined by the externalization of phosphatidylserine, the incorporation of propidium iodide, cell-cycle arrest at the sub-G0/G1 phase and DNA fragmentation into oligonucleosome-sized fragments. Despite the intrinsic variation that is detected in Leishmania spp, our results indicate that miltefosine causes apoptosis-like death in L. amazonensis promastigote cells using a similar process that is observed in Leishmania donovani.