The calpain inhibitor MDL28170 induces the expression of apoptotic markers in Leishmania amazonensis promastigotes.
BACKGROUND
Human 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.


RESULTS
In 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
The 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.
Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes (IMPG), Universidade Federal do Rio de Janeiro (UFRJ), Brasil.
Fernanda A
FA
Marinho
Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz (IOC), Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Brasil.
Keyla C S
KC
Gonçalves
Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes (IMPG), Universidade Federal do Rio de Janeiro (UFRJ), Brasil.
Simone S C
SS
Oliveira
Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes (IMPG), Universidade Federal do Rio de Janeiro (UFRJ), Brasil.
Diego S
DS
Gonçalves
Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes (IMPG), Universidade Federal do Rio de Janeiro (UFRJ), Brasil.
Filipe P
FP
Matteoli
Laboratório de Tecnologia em Cultura de Células, Centro Universitário Estadual da Zona Oeste (UEZO), Rio de Janeiro, Brasil.
Sergio H
SH
Seabra
Laboratório de Imunologia Molecular, Instituto de Biofísica Carlos Chagas Filho, UFRJ, Rio de Janeiro, Brasil.
Ana Carolina S
AC
Oliveira
Departamento de Imunologia, IMPG, UFRJ, Brasil.
Maria
M
Bellio
Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes (IMPG), Universidade Federal do Rio de Janeiro (UFRJ), Brasil.
Selma S
SS
Oliveira
Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes (IMPG), Universidade Federal do Rio de Janeiro (UFRJ), Brasil ; Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem (INBEB), UFRJ, Rio de Janeiro, Brasil.
Thaïs
T
Souto-Padrón
Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz (IOC), Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Brasil.
Claudia M
CM
d'Avila-Levy
Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes (IMPG), Universidade Federal do Rio de Janeiro (UFRJ), Brasil.
André L S
AL
Santos
Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes (IMPG), Universidade Federal do Rio de Janeiro (UFRJ), Brasil.
Marta H
MH
Branquinha
D017209:Apoptosis / Q000187:drug effects*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D053938:DNA Fragmentation / Q000187:drug effects*; D016054:DNA, Protozoan / Q000235:genetics / Q000378:metabolism*; D004151:Dipeptides / Q000494:pharmacology*; D016193:G1 Phase / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007891:Leishmania; D016773:Leishmaniasis, Cutaneous / Q000188:drug therapy / Q000201:enzymology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000235:genetics; D016192:Resting Phase, Cell Cycle / Q000187:drug effects
D015853:Cysteine Proteinase Inhibitors; D016054:DNA, Protozoan; D004151:Dipeptides; D015800:Protozoan Proteins; C058076:calpain inhibitor III