Trypanosoma cruzi proliferation and differentiation are blocked by topoisomerase II inhibitors

Abstract

Chagas' disease is a parasitic disease that affects more than 20 million individuals in Latin America (12). As yet, prophylaxis of the disease has been limited because of the lack of a vaccine or safe chemotherapy. Trypanosoma cruzi, the causative agent of this disease (4), displays a complex life cycle that involves two intermediary hosts (triatomines and mammals) and three differentiation stages: epimastigotes, which replicate in the insect vector; amastigotes, which replicate in mammalian cells; and the infective and nonreplicative trypomastigotes (11).The transformation of replicative forms into infective forms involves important morphogenetic changes. One of the most striking modifications occurs at the level of kinetoplast ultrastructure. The kinetoplast is that portion of the unique mitochondrion from members of the order Kinetoplastida which contains the mitochondrial DNA (kinetoplast DNA [K-DNA]). During cell proliferation, the K-DNA must replicate, while on transformation of amastigotes or epimastigotes into trypomastigotes, the K-DNA changes from a compact (bar) to a more relaxed (basket) form (11). This suggests that topoisomerases may play an important role in both T. cruzi proliferation and differentiation.Our recent work on T. cruzi metacyclogenesis (i.e., the transformation of epimastigotes into metacyclic trypomastigotes) resulted in the development of chemically defined in vitro differentiation conditions (1,8). In addition to allowing the production of metacyclic trypomastigotes and the study of the metacyclogenesis process, these conditions are very useful for testing the effects of drugs on T. cruzi differentiation.In this study we tested the effect of topoisomerase II inhibitors on T. cruzi metacyclogenesis. In addition, we also tested the effects of these inhibitors on T. cruzi proliferation and during the intracellular cycle of the parasite inside Parasites. The T. cruzi Dm28c clone was obtained and was kept in the laboratory as described previously (6). Epimastigotes were grown in liver infusion tryptose (LIT) medium (2). In vitro metacyclogenesis was performed as described elsewhere (8). Briefly, epimastigotes were harvested from LIT medium and incubated for 2 h in artificial triatomine urine (TAU) (190 mM NaCl, 17 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 8 mM phosphate buffer [pH 6.0]) and were then incubated in TAU supplemented with 10 mM L-proline (TAUP) for different times up to 96 h in the presence or absence of the drugs being tested. T. cruzi trypomastigotes of the Y strain (21) were obtained from the blood of albino mice at the peak of parasitemia and were isolated by differential centrifugation. Amastigote forms were obtained from the supernatant of a J774G-8 macrophage cell line, after 6 to 7 days after infection with bloodstream trypomastigotes (3). The proliferation of amastigotes in axenic culture (5 x 106 cells ml-') was performed in Warren medium at 29°C (10). In all cases, cells were counted in a Neubauer chamber.Treatment of heart muscle cell culture. Cultures that were 3 to 5 d...

In vitro effects of citral on Trypanosoma cruzi metacyclogenesis

“…Data from the literature have shown that the metacyclogenesis process of T. cruzi could be inhibited by the topoisomerase II inhibitor ofloxacin (Gonzales-Perdomo et al 1990), the antitubulin drug trifluralin (Bogitsh et al 1999), the proteasome inhibitor lactacystin (Cardoso et al 2008), metallo and cysteine-protease inhibitors (Bonaldo et al 1991) or mannose (Barbieri et al 1992). The data from the present paper showed that citral effectively blocked the metacyclogenesis of T. cruzi in vitro.…”

In Vitro and In Vivo Activities of 1,3,4-Thiadiazole-2-Arylhydrazone Derivatives of Megazol against Trypanosoma cruzi

“…This structure represents a potential target in T. cruzi for different drugs (28,41). Examples of such drugs are topoisomerase inhibitors (18,20), aromatic diamidines and reversed amidines (37,40), and the putrescine analogue 1,4-diamino-2-butanone (25). The SEM analysis revealed that the treatment induced severe morphological changes in trypomastigotes (Fig.…”

A Critical Review on Chagas Disease Chemotherapy

“…Several inhibitors of bacterial DNA topoisomerase II showed activity against T. cruzi, inhibiting both proliferation and differentiation processes, and causing damage to kinetoplast and/or the nucleus of epimastigotes (Kerschmann et al 1989, Gonzales-Perdomo et al 1990), suggesting that both organelles could be the targets of the drugs. Camptothecin, inhibitor of eukaryotic DNA topoisomerase I, induced cleavage of nuclear and mitochondrial DNA in T. cruzi (Bodley & Shapiro 1995).…”