Abstract
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.
Highlights
According to the World Health Organization [1], cutaneous and visceral leishmaniases are included in the list of neglected tropical diseases, being considered a major global threat for health
Human cutaneous leishmaniasis is caused by distinct species in the Old and New World, including Leishmania amazonensis in the latter
The calpain inhibitor MDL28170 was added to L. amazonensis promastigote forms in different concentrations, and the cellular growth was compared with a control culture after 72 h
Summary
According to the World Health Organization [1], cutaneous and visceral leishmaniases are included in the list of neglected tropical diseases, being considered a major global threat for health. Human cutaneous leishmaniasis is caused by distinct species in the Old and New World, including Leishmania amazonensis in the latter. This trypanosomatid is associated with all clinical forms of the disease, which includes localized, mucocutaneous and diffuse cutaneous leishmaniasis [2], and is commonly found in the Amazon region, encompassing many Latin American countries [3]. 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
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