Elsevier

Acta Tropica

Volume 211, November 2020, 105653
Acta Tropica

Antiproliferative activity of the dibenzylideneacetone derivate (E)-3-ethyl-4-(4-nitrophenyl)but‑3-en-2-one in Trypanosoma cruzi

https://doi.org/10.1016/j.actatropica.2020.105653Get rights and content

Highlights

  • Dibenzylidenoacetones shows important in vitro activity against trypanosoma cruzi.

  • The dibenzylidenoacetone A11K3 is active against all three forms of T. cruzi.

  • A11K3 causes death parasite due alterations in membrane and mitochondria.

Abstract

Chagas disease is one of the most prevalent neglected diseases in the world. The illness is caused by Trypanosoma cruzi, a protozoan parasite with a complex life cycle and three morphologically distinct developmental stages. Nowadays, the only treatment is based on two nitro-derivative drugs, benznidazole and nifurtimox, which cause serious side effects. Since the treatment is limited, the search for new treatment options for patients with Chagas disease is highly necessary. In this study we analyzed the substance A11K3, a dibenzylideneacetone (DBA). DBAs have an acyclic dienone attached to aryl groups in both β-positions and studies have shown that they have biological activity against tumors cells, bacteria, and protozoa such as T. cruzi and Leishmania spp. Here we show that A11K3 is active against all three T. cruzi evolutionary forms: the epimastigote (IC50 = 3.3 ± 0.8), the trypomastigote (EC50 = 24 ± 4.3) and the intracellular amastigote (IC50 = 9.3 ± 0.5 µM). A cytotoxicity assay in LLCMK2 cells showed a CC50 of 239.2 ± 15.7 µM giving a selectivity index (CC50/IC50) of 72.7 for epimastigotes, 9.9 for trypomastigotes and 25.9 for intracellular amastigotes. Morphological and ultrastructural analysis of the parasites treated with A11K3 by TEM and SEM revealed alterations in the Golgi complex, mitochondria, plasma membrane and cell body, with an increase of autophagic vacuoles and lipid bodies. Biochemical assays of A11K3-treated T. cruzi showed an increase of ROS, plasma membrane ruptures, lipid peroxidation, mitochondrial membrane depolarization with a decrease in ATP and accumulation of autophagic vacuoles. The results lead to the hypothesis that A11K3 causes death of the protozoan through events such as plasma membrane and mitochondrial alterations and autophagy, characteristic of cell collapse.

Introduction

Chagas disease is a neglected tropical disease caused by the protozoan Trypanosoma cruzi, affecting 6–7 million people worldwide (WHO, 2019). This protozoan parasite displays three distinct evolutionary relevant forms: the epimastigote, found inside the hematophagous insect; the trypomastigote, which is the infective form found in the insect vector and in the blood circulation of the mammalian host; and the amastigote, found intracellularly in tissues of the mammalian host. The epimastigote and amastigote are the replicative forms of the parasite (Alves et al., 2007; Nogueira et al., 2007).

Infection by T. cruzi has two clinical phases: the acute phase characterized by high parasitemia; and the chronic phase, which is long and progressive whereby symptoms can manifest after some years. The treatment of Chagas disease is performed with two drugs, nifurtimox or benznidazole. However, they have several side effects and limited therapeutic potential (Coura, 2009; Izumi et al., 2011).

Several studies have shown that synthetic compounds have great potential for the treatment of Chagas’ disease as thiazoles, β-lapachone, β-carbolines, quinoxalines (Cogo et al., 2015; de Oliveira Filho et al., 2017; Garavaglia et al., 2018; Moreno-Viguri and Pérez-Silanes, 2013; Valdez et al., 2012). 1,5-Diarylpentanoid dibenzylideneacetone (DBA) and their derivatives are compounds that have an acyclic dienone attached to aryl groups in both β-positions. These compounds have demonstrated antiproliferative, anti-inflammatory and apoptotic effects in cancer cell lines (Bhandarkar et al., 2008; Prasad et al., 2011; Ud Din et al., 2014; Yu et al., 2013). Others studies have reported the anti-leishmanial and anti-trypanosomal effects of the DBAs derivatives (Garcia et al., 2017; Lazarin-Bidóia et al., 2016). Considering that the available chemotherapy is inefficient for patients, research is a need in search of new therapies (Wilkinson and Kelly, 2009). Based on the context, the aim of this study is to elucidate the activity and the main alterations induced by dibenzylidenoacetone derivate (E)−3-ethyl-4-(4-nitrophenyl)but-3-en-2-one (A11K3) against epimastigotes, trypomastigotes and intracellular amastigotes of T. cruzi.

Section snippets

Chemicals

The following chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA): carbonyl cyanide m-chlorophenylhydrazone (CCCP), rhodamine 123 (Rh123), dansylcadaverine (MDC), Nile red, digitonin, methylphenazinium methyl sulfate (PMS), dihydrochlorofluorescein (H2DCFDA), wortmannin, potassium cyanate (KCN) and dimethyl sulfoxide (DMSO). LIT (liver infusion yeast extract), hemin and folic acid were acquired from Sigma-Aldrich (St. Louis, MO, USA). The

Preparation of the derivate A11K3

The reaction of p-nitrobenzaldehyde (A11) with 2-pentanone (K3), using gaseous HCl as a catalyst, as outlined in Fig. 2, was performed at room temperature by stirring the reaction mixture and passing the dry gaseous HCl over the reaction until the mixture turned white-yellow (Fig. 2).

Activity of A11K3 against T. cruzi epimastigotes, trypomastigotes and intracellular amastigotes

In the present study, we analyzed the activity of a dibenzylideneacetone, A11K3, against the three developmental forms of T. cruzi. The compound showed an IC50/96h of 3.3 ± 0.8 µM against epimastigotes, an EC50/24 h

Discussion

In our recent studies on the synthesis of curcumin analogues, or diaryalkadienones, we have performed many different reactions of aldehydes (An) with ketones (Km) in various conditions to produce the aldols intermediaries following its condensation on pot (Din et al., 2018). The reaction of p-nitrobenzaldehyde (A11) with 2-pentanone (K3) occurs via the thermodynamic enolate giving good regiochemistry control, yielding almost 100% of A11K3 without a detectable amount of aldol condensation via

Conclusion

This study with a DBA derivate A11K3 has demonstrated that the compound is efficient against epimastigotes, trypomastigotes and intracellular amastigotes of T. cruzi. The biochemical assays are performed with epimastigotes and intracellular amastigotes because are forms replicative of T. cruzi. In addition, the amastigotes are forms found during chronic phase of Chagas disease, collaborating for the next steps of the study with A11K3.

The results suggest that A11K3 has less toxic for mammalian

CRediT authorship contribution statement

Jéssica Carreira de Paula: Conceptualization, Data curation, Formal analysis, Methodology, Investigation, Writing - original draft. Amanda Beatriz Kawano Bakoshi: Validation, Visualization, Methodology, Writing - review & editing. Danielle Lazarin-Bidóia: Validation, Visualization, Methodology, Writing - review & editing. Zia Ud Din: Methodology, Validation. Edson Rodrigues-Filho: Methodology, Validation. Tania Ueda-Nakamura: Funding acquisition, Project administration, Supervision, Writing -

Declaration of Competing Interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This study was supported by the Coordination of Improvement of Higher Level Personnel (Capes – Brazil), National Council for Scientific and Technological Development and Sciences without Borders Program (CNPq – Brazil), Araucaria Foundation (Brazil), Financier of Studies and Projects (Finep - Brazil) and COMCAP – UEM. The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.

References (55)

  • L. Monzote et al.

    Experimental parasitology essential oil from Chenopodium ambrosioides and main components : activity against Leishmania , their mitochondria and other microorganisms

    Exp. Parasitol.

    (2014)
  • S.B. Mukherjee et al.

    Increase in cytosolic Ca2+ levels through the activation of non-selective cation channels induced by oxidative stress causes mitochondrial depolarization leading to apoptosis-like death in Leishmania donovani promastigotes

    J. Biol. Chem.

    (2002)
  • N.F.S. Nogueira et al.

    Trypanosoma cruzi: involvement of glycoinositolphospholipids in the attachment to the luminal midgut surface of Rhodnius prolixus

    Exp. Parasitol.

    (2007)
  • S. Patterson et al.

    Nitro drugs for the treatment of trypanosomatid diseases: past, present, and future prospects

    Trends Parasitol.

    (2014)
  • Z. Ud Din et al.

    Unsymmetrical 1,5-diaryl-3-oxo-1,4-pentadienyls and their evaluation as antiparasitic agents

    Bioorg. Med. Chem.

    (2014)
  • L.D. Zorova et al.

    Mitochondrial membrane potential

    Anal. Biochem.

    (2018)
  • S.S. Bhandarkar et al.

    Cancer therapy : preclinical ris (Dibenzylideneacetone ) Dipalladium , a N -Myristoyltransferase-1 Inhibitor, Is effective against melanoma growth In vitro and In vivo

    Clin. Cancer Res.

    (2008)
  • E.F.C. Blommaart et al.

    The phosphatidylinositol 3-Kinase inhibitors Wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes

    Eur. J. Biochem.

    (1997)
  • M. Bogacz et al.

    Tryparedoxin peroxidase-deficiency commits trypanosomes to ferroptosis-type cell death

    Elife

    (2018)
  • J. Boren et al.

    Apoptosis-induced mitochondrial dysfunction causes cytoplasmic lipid droplet formation

    Cell Death Differ.

    (2012)
  • P.T. Bozza et al.

    Induction of autophagy correlates with increased parasite load of Leishmania amazonensis in BALB/c but not C57BL/6 macrophages

    Microbes Infect.

    (2008)
  • Z. Brener

    Therapeutic activity and criterion of cure on mice experimentally infected with Trypanosoma cruzi

    Rev. Inst. Med. Trop. São Paulo

    (1962)
  • E.A. Britta et al.

    Cell death and ultrastructural alterations in Leishmania amazonensis caused by new compound 4-Nitrobenzaldehyde thiosemicarbazone derived from S-limonene

    BMC Microbiol.

    (2014)
  • C.F. Brooks et al.

    Autophagy protein Atg3 is essential for maintaining mitochondrial integrity and for normal intracellular development of Toxoplasma gondii tachyzoites

    Plos Pathog.

    (2011)
  • J.R. Coura

    Present situation and new strategies for Chagas disease chemotherapy: a proposal

    Mem. Inst. Oswaldo Cruz

    (2009)
  • S.T. De Macedo-Silva et al.

    In vitro activity of the antifungal azoles itraconazole and posaconazole against Leishmania amazonensis

    PLoS ONE

    (2013)
  • J.C. Engel et al.

    Cysteine protease inhibitors alter Golgi complex ultrastructure and function in Trypanosoma cruzi

    J. Cell Sci.

    (1998)
  • Cited by (5)

    View full text