Miltefosine induces programmed cell death in Leishmania amazonensis promastigotes

Marinho, Fernanda de Aquino; Goncalves, Keyla Cristiny da Silva; Oliveira, Selma Soares de; Oliveira, Ana de Siqueira Couto de Carolina; Bellio, Maria; d’Avila-Levy, Claudia M.; Santos, André Luis Souza dos; Branquinha, Marta H.

doi:10.1590/s0074-02762011000400021

Cited by 45 publications

(24 citation statements)

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“…Non-treated cells were Annexin-V- and PI-negative (Figure 4), and the same result was seen in cells treated only with DMSO, the vehicle of drug (data not shown), which confirms the viability of cells in these conditions. Alternatively, promastigotes treated with miltefosine were used as a positive control in this experiment [23], [24]. After 24 h of incubation with miltefosine at 40 µM, 1.02% of cells were apoptotic-like (Annexin-V-positive and PI-negative) and 81.8% of cells were already in late apoptotic-like phase or necrosis (Annexin-V-positive and PI-positive) (Figure 4).…”

Section: Resultsmentioning

confidence: 99%

“…The intensity of labeling of Annexin-V conjugated to Alexa-Fluor was recorded in a FACSCalibur (BD Biosciences) flow cytometer and analyzed with Summit software, and the percentage of positive cells was assessed for each histogram. Miltefosine, an established inducer of apoptosis in L. amazonensis promastigotes (at 40 µM for 24 h) was used as a positive control [23], [24].…”

Section: Methodsmentioning

confidence: 99%

“…A total of 10,000 events were acquired in the region previously established as that corresponding to the parasites. Miltefosine (at 40 µM for 24 h) served as the positive control [23], [24].…”

Section: Methodsmentioning

confidence: 99%

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The Calpain Inhibitor MDL28170 Induces the Expression of Apoptotic Markers in Leishmania amazonensis Promastigotes

et al. 2014

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BackgroundHuman cutaneous leishmaniasis is caused by distinct species, including Leishmania amazonensis. Treatment of cutaneous leishmaniasis is far from satisfactory due to increases in drug resistance and relapses, and toxicity of compounds to the host. As a consequence for this situation, the development of new leishmanicidal drugs and the search of new targets in the parasite biology are important goals.Methodology/Principal FindingsIn this study, we investigated the mechanism of death pathway induced by the calpain inhibitor MDL28170 on Leishmania amazonensis promastigote forms. The combined use of different techniques was applied to contemplate this goal. MDL28170 treatment with IC50 (15 µM) and two times the IC50 doses induced loss of parasite viability, as verified by resazurin assay, as well as depolarization of the mitochondrial membrane, which was quantified by JC-1 staining. Scanning and transmission electron microscopic images revealed drastic alterations on the parasite morphology, some of them resembling apoptotic-like death, including cell shrinking, surface membrane blebs and altered chromatin condensation pattern. The lipid rearrangement of the plasma membrane was detected by Annexin-V labeling. The inhibitor also induced a significant increase in the proportion of cells in the sub-G0/G1 phase, as quantified by propidium iodide staining, as well as genomic DNA fragmentation, detected by TUNEL assay. In cells treated with MDL28170 at two times the IC50 dose, it was also possible to observe an oligonucleossomal DNA fragmentation by agarose gel electrophoresis.Conclusions/SignificanceThe data presented in the current study suggest that MDL28170 induces apoptotic marker expression in promastigotes of L. amazonensis. Altogether, the results described in the present work not only provide a rationale for further exploration of the mechanism of action of calpain inhibitors against trypanosomatids, but may also widen the investigation of the potential clinical utility of calpain inhibitors in the chemotherapy of leishmaniases.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The Calpain Inhibitor MDL28170 Induces the Expression of Apoptotic Markers in Leishmania amazonensis Promastigotes

et al. 2014

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“…Miltefosine is an inhibitor of Akt in Leishmania (15), and perifosine also inhibits Akt protein in human cancer cells (16). These facts lead us to believe that perifosine has the same Akt-inhibiting capacity in Leishmania, which leads to the induction of an apoptosis-like cell death that is known to occur with miltefosine in L. amazonensis (17) and L. donovani (18).…”

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confidence: 99%

Perifosine Mechanisms of Action in Leishmania Species

López-Arencibia

Martín-Navarro

Sifaoui

et al. 2017

Antimicrob Agents Chemother

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Here the mechanism by which perifosine induced cell death in Leishmania donovani and Leishmania amazonensis is described. The drug reduced Leishmania mitochondrial membrane potential and decreased cellular ATP levels while increasing phosphatidylserine externalization. Perifosine did not increase membrane permeabilization. We also found that the drug inhibited the phosphorylation of Akt in the parasites. These results highlight the potential use of perifosine as an alternative to miltefosine against Leishmania.

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“…It is known that both miltefosine and Sb III can induce programmed cell death (PCD) or necrosis (43)(44)(45)(46). We used propidium iodide (PI) and annexin V to stain promastigotes after miltefosine or Sb III exposure.…”

Section: (D) Ultrastructure Of the Flagellar Pocket Of L Donovani Prmentioning

confidence: 99%

A Telomeric Cluster of Antimony Resistance Genes on Chromosome 34 of Leishmania infantum

Nevado

Bifeld

Höhn

et al. 2016

Antimicrob Agents Chemother

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The mechanisms underlying the drug resistance of Leishmania spp. are manifold and not completely identified. Apart from the highly conserved multidrug resistance gene family known from higher eukaryotes, Leishmania spp. also possess genus-specific resistance marker genes. One of them, ARM58, was first identified in Leishmania braziliensis using a functional cloning approach, and its domain structure was characterized in L. infantum. Here we report that L. infantum ARM58 is part of a gene cluster at the telomeric end of chromosome 34 also comprising the neighboring genes ARM56 and HSP23. We show that overexpression of all three genes can confer antimony resistance to intracellular amastigotes. Upon overexpression in L. donovani, ARM58 and ARM56 are secreted via exosomes, suggesting a scavenger/secretion mechanism of action. Using a combination of functional cloning and next-generation sequencing, we found that the gene cluster was selected only under antimonyl tartrate challenge and weakly under Cu 2؉ challenge but not under sodium arsenite, Cd 2؉ , or miltefosine challenge. The selective advantage is less pronounced in intracellular amastigotes treated with the sodium stibogluconate, possibly due to the known macrophage-stimulatory activity of this drug, against which these resistance markers may not be active. Our data point to the specificity of these three genes for antimony resistance. P arasitic protozoa of the genus Leishmania, order Trypanosomatida, are responsible for the various clinical manifestations of leishmaniasis. The disease is transmitted by the bite of sandflies, with 2 million new infections occurring per year, and occurs in 98 countries on five continents (1). Leishmania spp. exist in two morphologically distinct life cycle stages. In the transmitting sandflies, the flagellated and elongated promastigotes proliferate attached to the epithelium of the digestive tract until they reach a high density. A switch in the surface molecule composition then causes detachment, and the so-called metacyclic promastigotes are free to invade a mammalian host when the sandfly takes a blood meal. Inside the mammalian skin, the parasites are quickly taken up by antigen-presenting cells, such as dendritic cells, neutrophilic granulocytes, and macrophages, and establish themselves in these cells as small, ovoid, aflagellated amastigotes. The resulting destruction of infected macrophages causes inflammatory immune responses and the concomitant immune pathologies.Depending on the infecting species and on the host's immune status, Leishmania-related pathologies range from localized, ulcerating skin lesions (cutaneous leishmaniasis [CL]) and diffuse cutaneous lesion formation (diffuse cutaneous leishmaniasis [DCL]) to mucocutaneous leishmaniasis (MCL) and, lastly, a generalized infection known as kala azar (visceral leishmaniasis [VL]). VL is invariably lethal in untreated cases, while MCL can also have lethal outcomes due to secondary infections of the nasopharyngeal area.As there is no vaccine against Leishmania a...

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Miltefosine induces programmed cell death in Leishmania amazonensis promastigotes

Cited by 45 publications

References 13 publications

The Calpain Inhibitor MDL28170 Induces the Expression of Apoptotic Markers in Leishmania amazonensis Promastigotes

The Calpain Inhibitor MDL28170 Induces the Expression of Apoptotic Markers in Leishmania amazonensis Promastigotes

Perifosine Mechanisms of Action in Leishmania Species

A Telomeric Cluster of Antimony Resistance Genes on Chromosome 34 of Leishmania infantum

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