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

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.

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“…However, other phospholipid classes which participate in apoptosis, like phosphatidylethanol sphingomyelin and phosphatidyl choline, can also enable annexin V staining, and therefore annexin V binding is used as an early indicator of apoptosis by many other investigators (35,50). Our results of annexin V-FITC and PI indicated necrosis as shift from the UR to the UL quadrant of synthesized-Ag NP-treated parasites.…”

“…The histogram and images are representative of three independent experiments. A total of 10,000 events from each sample were acquired to ensure adequate data (35).…”

Ruthenium-Clotrimazole complex has significant efficacy in the murine model of cutaneous leishmaniasis

“…Depolarization of mitochondria transmembrane potential ( Δψm ) is an initial biochemical facet implying activation of the intrinsic apoptotic pathway, as the initial triggering signal of apoptosis (Marinho et al, 2014). In addition, it has been proven to be an important feature for cell death in protozoa parasites such as Leishmania (Sen et al, 2004).…”

Expression of calpain-like proteins and effects of calpain inhibitors on the growth rate of Angomonas deanei wild type and aposymbiotic strains

“…Further studies showed that MDL28170 was able to inhibit the interaction of trypomastigotes with macrophages and intracellular survival [ 28 ], besides interfering in the parasite adhesion to the insect midgut and in the differentiation process of epimastigotes into metacyclic trypomastigotes [ 18 ]. Interestingly, in L. amazonensis , MDL28170 was also shown to induce the expression of apoptotic markers [ 29 ].…”