Primary targets of the sesquiterpene lactone deoxymikanolide on Trypanosoma cruzi

Abstract

Background: Deoxymikanolide is a sesquiterpene lactone isolated from Mikania micrantha and M. variifolia which, has previously demonstrated in vitro activity on Trypanosoma cruzi and in vivo activity on an infected mouse model.

Purpose: Based on these promising findings, the aim of this study was to investigate the mechanism of action of this compound on different parasite targets.

Methods: The interaction of deoxymikanolide with hemin was examined under reducing and non- reducing conditions by measuring modifications in the Soret absorption band of hemin; the thiol interaction was determined spectrophotometrically through its reaction with 5,5'-dithiobis-2-nitrobenzoate in the presence of glutathione; activity on the parasite antioxidant system was evaluated by measuring the activity of the superoxide dismutase and trypanothione reductase enzymes, together with the intracellular oxidative state by flow cytometry. Superoxide dismutase and trypanothione reductase activities were spectrophotometrically tested. Cell viability, phosphatidylserine exposure and mitochondrial membrane potential were assessed by means of propidium iodide, annexin-V and rhodamine 123 staining, respectively; sterols were qualitatively and quantitatively tested by TLC; ultrastructural changes were analyzed by transmission electron microscopy. Autophagic cells were detected by staining with monodansylcadaverine.

Results: Deoxymikanolide decreased the number of reduced thiol groups within the parasites, which led to their subsequent vulnerability to oxidative stress. Treatment of the parasites with the compound produced a depolarization of the mitochondrial membrane even though the plasma membrane permeabilization was not affected. Deoxymikanolide did not affect the intracellular redox state and so the mitochondrial dysfunction produced by this compound could not be attributed to ROS generation. The antioxidant defense system was affected by deoxymikanolide at twenty four hours of treatment, when both an increased oxidative stress and decreased activity of superoxide dismutase and trypanothione reductase (40 and 60% respectively) were observed. Both the oxidative stress and mitochondrial dysfunction induce parasite death by apoptosis and autophagy.

Conclusion: Based on our results, deoxymikanolide would exert its anti-T cruzi activity as a strong thiol blocking agent and by producing mitochondrial dysfunction.

Keywords: DCF: Dichlorofluorescein; DCIP: 2,6-dichlorophenolindophenol, DMSO: dimethyl sulfoxide, DTNB: 5,5′-dithiobis-2-nitrobenzoate, GSH: glutathione, PBS: phosphate buffered saline, PI: propidium iodide, Rh123: rhodamine 123, ROS: reactive oxygen species, SCR: succinate cytochrome c reductase, SOD: superoxide dismutase, TryR: trypanothione reductase; Deoxymikanolide; H2DCFDA: 2′,7′-dichlorodihydrofluorescein diacetate; Mitochondrial dysfunction; Oxidative stress; Sesquiterpene lactone; Trypanosoma cruzi; Ultraestructural damage.