Naphthoimidazoles promote different death phenotypes in Trypanosoma cruzi

Abstract

In a screening of 65 derivatives of natural quinones using bloodstream trypomastigotes of Trypanosoma cruzi, the 3 naphthoimidazoles derived from beta-lapachone - N1, N2 and N3--were selected as the most active. Investigation of their mode of action led to the characterization of mitochondrion, reservosomes and DNA as their main targets, and stimulated further studies on death pathways. Ultrastructural analysis revealed both autophagic (autophagosomes) and apoptotic-like (membrane blebbing) phenotypes. Flow cytometry analysis showed, in N2-treated trypomastigotes, a small increase of phosphatidylserine exposure, and a large increase in the percentage of necrosis, caused by N1 or N2. These death phenotypes were not detected in treated epimastigotes. The strong increase in labelling of monodansyl cadaverine, the inhibition of the death process by wortmannin or 3-methyladenine, the overexpression of ATG genes in treated epimastigotes, together with ultrastructural evidence point to autophagy as the predominant phenotype induced by the naphthoimidazoles. However, there are other pathways occurring concomitantly with variable intensities, justifying the need to detail the molecular features involved.

Autophagy in protists

“…131 Pre-incubation with the autophagic inhibitors wortmannin and 3-methyladenine (3-MA) completely abolish the effects of N1, N2 and N3, pointing to autophagic cell death as a consequence of this treatment. 130 In treated epimastigote forms, extensive ER profiles surround structures, especially reservosomes, proving a close contact between the membranes of both organelles ( Fig. 9 and Table 1).…”

“…For T. brucei, the example is the killing of parasites as a result of administration of DHA or neuropeptides, 120,121 while for T. cruzi the same was achieved with lysophospholipid analogues and naphtoimidazoles. 127,130 However, similar data regarding the induction of autophagic cell death of Leishmania parasites by administration of compounds are not available.…”

“…128,129 Frequently, treatment with these three compounds induces morphological autophagic characteristics in the parasites such as the occurrence of concentric membrane structures in the cytosol and an increase in the number of autophagosome-like bodies, observations reinforced by the strong increase of monodansylcadaverine labeling. 130 The effect of the naphthoimidazoles on both forms of T. cruzi is blocked by E64 and calpain I inhibitor, 129 suggesting the participation of calpain in the autophagic process as in mammalian cells. 131 Pre-incubation with the autophagic inhibitors wortmannin and 3-methyladenine (3-MA) completely abolish the effects of N1, N2 and N3, pointing to autophagic cell death as a consequence of this treatment.…”

“…RT-PCR data indicate the participation of ATG3, ATG4, ATG7 and ATG8 in the autophagic process in epimastigotes treated with naphthoimidazoles, 130 although the detailed pathway and proteins involved in the mechanisms of cell death require further investigation.…”

Cell death offers exceptional cellular and molecular windows for pharmacological interventions in protozoan parasites

“…The autophagy -apoptosis -necrosis continuum depends on parasite insult, response stringency and type, and cellular constituent and signaling pathway influences. Interestingly, autophagic (autophagosomes), apoptoticlike (membrane blebbing) phenotypes, and necrosis have been reported in Naphthoimidazoles-treated Trypanosoma cruzi [1]. It has been demonstrated that an intense cross-talking between autophagy, apoptosis, and necrosis in the protozoa in response to diverse drug stimuli that cover the same pathway of the previous processes [2].…”