Isoxazole 35-a Causes Apoptosis-like Programmed Cell Death in Leishmania amazonensis Promastigotes

Vol. 2, 2019. - 114314
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Introduction: Leishmaniasis is a vector borne parasitic disease that affects millions of people worldwide. Despite this, the current chemotherapy is still limited and exhibits toxicity and several side effects; thus, the search for new compounds with leishmanicidal activity remains a necessity. Isoxazoles are an important class of heterocycles, used in the pharmaceuticals and therapeutics areas. Therefore, the goal of this study was to evaluate the production of reactive oxygen species (ROS), the alterations in mitochondrial membrane potential (ΔΨm), cell cycle arrest and changes in cell volume in Leishmania amazonensis promastigotes induced by the synthetic compound 4-[((4-fluorophenyl)amino)methyl]-5-(4-nitrophenyl)-3-[N’-(pyridin-2-ylmethylene)hydrazinecarbonyl]-isoxazole (35-a). Methods: promastigotes were treated with IC50 (12.7 µM) and 2xIC50 (25.4 µM) of 35-a for 24 h at 25 ºC. For the detection of ROS, promastigotes were incubated with 10 µM H2DCFDA and the fluorescence was determined using a fluorescence microplate reader (Victor X3; PerkinElmer). For ΔΨm measurement, parasites were treated at the same concentrations described, followed by incubation with 5 µg/mL rhodamine 123 (Rh123). As for the cell cycle analysis, treated promastigotes were fixed in 70% cold methanol–distilled water and then incubated with 20 µL propidium iodide-RNase A. The cell volume was determined on treated promastigotes ressuspended in phosphate-buffered saline. The ΔΨm assay, cell cycle analysis and cell volume determination were carried out using a BD FACSCalibur™ flow cytometer equipped with CellQuest software. Results: treatment with isoxazole 35-a caused a significant increase in ROS production of 168% and 319% in promastigotes treated with IC50 and 2xIC50 of 35-a, respectively, compared with untreated cells. Mitochondria is the main source of free radicals as ROS, and its increased production may have led to an alteration in ΔΨm, observed by the decrease (17%) of Rh123 fluorescence intensity in promastigotes treated with 2xIC50 of 35-a, compared with control group. Elevated intracellular levels of ROS cause oxidative stress and damage to proteins, lipids and DNA. Cell cycle analysis showed that 35-a induces cell cycle arrest in the sub-G0/G1 phase of treated parasites; the significant increase in the cell population of this phase represents DNA fragmentation, which is associated with apoptosis-like programmed cell death (PCD). Results also showed a decrease in the G2/M cell population (phase of DNA duplication) of 43,9% and 72,9% using IC50 and 2xIC50 of 35-a, respectively. A significant reduction in cell volume of treated parasites was observed by flow cytometry, of 23,8% and 35% in promastigotes treated with IC50 and 2xIC50 of 35-a, respectively, compared with the control group, which is also characteristic of apoptosis-like PCD. Conclusions: our results demonstrated that the isoxazole 35-a generates oxidative stress in L. amazonensis promastigotes, leading to biochemical and morphological alterations that are characteristic of apoptosis-like PCD.

Instituições
  • 1 Universidade Estadual de Maringá
Eixo Temático
  • Biotecnologia Microbiana
Palavras-chave
Leishmania amazonensis
isoxazole
cell death
Oxidative stress