11 research outputs found
The Effectiveness of Natural Diarylheptanoids against <i>Trypanosoma cruzi</i>: Cytotoxicity, Ultrastructural Alterations and Molecular Modeling Studies
<div><p>Curcumin (CUR) is the major constituent of the rhizomes of <i>Curcuma longa</i> and has been widely investigated for its chemotherapeutic properties. The well-known activity of CUR against <i>Leishmania sp</i>., <i>Trypanosoma brucei</i> and <i>Plasmodium falciparum</i> led us to investigate its activity against <i>Trypanosoma cruzi</i>. In this work, we tested the cytotoxic effects of CUR and other natural curcuminoids on different forms of <i>T</i>. <i>cruzi</i>, as well as the ultrastructural changes induced in epimastigote form of the parasite. CUR was verified as the curcuminoid with more significant trypanocidal properties (IC<sub>50</sub> 10.13 μM on epimastigotes). Demethoxycurcumin (DMC) was equipotent to CUR (IC<sub>50</sub> 11.07 μM), but bisdemethoxycurcumin (BDMC) was less active (IC<sub>50</sub> 45.33 μM) and cyclocurcumin (CC) was inactive. In the experiment with infected murine peritoneal macrophages all diarylheptanoids were more active than the control in the inhibition of the trypomastigotes release<b>.</b> The electron microscopy images showed ultrastructural changes associated with the cytoskeleton of the parasite, indicating tubulin as possible target of CUR in <i>T</i>. <i>cruzi</i>. The results obtained by flow cytometry analysis of DNA content of the parasites treated with natural curcuminoids suggested a mechanism of action on microtubules related to the paclitaxel`s mode of action. To better understand the mechanism of action highlighted by electron microscopy and flow cytometry experiments we performed the molecular docking of natural curcuminoids on tubulin of <i>T</i>. <i>cruzi</i> in a homology model and the results obtained showed that the observed interactions are in accordance with the IC<sub>50</sub> values found, since there CUR and DMC perform similar interactions at the binding site on tubulin while BDMC do not realize a hydrogen bond with Lys163 residue due to the absence of methoxyl groups. These results indicate that trypanocidal properties of CUR may be related to the cytoskeletal alterations.</p></div
ChemPLP scores and hydrogen bond distances for curcumin derivatives from docking into <i>T</i>. <i>cruzi</i> tubulin.
<p>ChemPLP scores and hydrogen bond distances for curcumin derivatives from docking into <i>T</i>. <i>cruzi</i> tubulin.</p
Scanning electron microscopy of <i>T</i>. <i>cruzi</i> epimastigotes.
<p>Untreated control cells (A) displayed the usual elongated morphology with smooth cell surface. Parasites incubated with 10.13 μM curcumin for 24 h (B-D) presented reduced cell volume (B) as well as cell body rounding with multiple longitudinal invaginations involving the anterior portion of the parasite (C,D). Some cells displayed multiple shortened flagella (C, arrows). The protrusion of flagellar membrane was detected (D, arrows).</p
Structures of the main chemical constituents from <i>Curcuma longa</i>.
<p>Structures of the main chemical constituents from <i>Curcuma longa</i>.</p
Superposition of best qualified poses of CUR (Entry A), DMC (Entry B) and BDMC (Entry C) after docking into <i>T</i>. <i>cruzi</i> tubulin.
<p>Hydrogen atoms were omitted for clarity; carbon atoms in green: tubulin; yellow: CUR; cyan: DMC; magenta: BDMC. (image generated with PyMOL, DeLano Scientific LLC)</p
Scanning electron microscopy showing <i>T</i>. <i>cruzi</i> epimastigotes incubated with curcumin for 48 h (A) and transmission electron microscopy of parasites treated for 72 h (B-E).
<p>Curcumin at 10.13 μM induced the formation of flagellar protrusions (A, arrowheads). The paraflagellar rod (B White arrowhead) is partially disorganized (B, black arrowhead). (E). Besides intraflagellar disorganization (C, arrowhead) and curcumin-treated cells displayed flagellar membrane detachment (C, arrows). Such disorganization was observed in flagella (F) within the flagellar pocket lumen (D, arrowhead) and may be associated to basal body (E, black arrowhead) alterations, which included ectopic microtubules duplets (white arrowhead).</p
IC<sub>50</sub> values for epimastigotes of <i>T</i>. <i>cruzi</i> (Dm28c strain) and LD<sub>50</sub> for two cell lines.
<p>IC<sub>50</sub> values for epimastigotes of <i>T</i>. <i>cruzi</i> (Dm28c strain) and LD<sub>50</sub> for two cell lines.</p
Percentage of cells in different phases of cell cycle.
<p>Cells were treated with CUR and DMC at 10, 50 and 100 μM concentrations and with 100 μM BDMC. Paclitaxel (20 μM) was employed as positive control, and DMSO 0.02% as negative control. Results are the mean of the triplicates with standard deviation represented by error bars. Statistical significance relative to control * <i>P</i><0.05, ** <i>P</i><0.01 and *** <i>P</i><0.001. The data shown were obtained from three independent experiments.</p
Murine peritoneal macrophages were infected with 10<sup>5</sup> <i>T</i>. <i>cruzi</i> trypomastigotes (Dm28c strain).
<p>Parasite cultures were treated with 100 μM of benznidazole, CUR, DMC, BDMC and CC. After 7 days, the number of released trypomastigotes in the culture medium were determined. The data shown were obtained from three independent experiments. Cultures were compared using unpaired T-Student test (Graph Pad Prism). *P<0.05.</p
Synthesis of CUR and BDMC via acetylacetone-boron enolate.
<p>Synthesis of CUR and BDMC via acetylacetone-boron enolate.</p