Apoptosis-Like Cell Death Upon Kinetoplastid Induction By

Chiboub et al. Parasites Vectors (2021) 14:198 https://doi.org/10.1186/s13071-021-04693-7 Parasites & Vectors RESEARCH Open Access Apoptosis-like cell death upon kinetoplastid induction by compounds isolated from the brown algae Dictyota spiralis Olfa Chiboub1,2,3,4* , Ines Sifaoui1,2,5, Manef Abderrabba4, Mondher Mejri4, José J. Fernández3,6, Ana R. Díaz‑Marrero3, Jacob Lorenzo‑Morales1,2,5 and José E. Piñero1,2,5 Abstract Background: The in vitro activity of the brown seaweed Dictyota spiralis against both Leishmania amazonensis and Trypanosoma cruzi was evaluated in a previous study. Processing by bio‑guided fractionation resulted in the isolation of three active compounds, classifed as diterpenes. In the present study, we performed several assays to detect clini‑ cal features associated to cell death in L. amazonensis and T. cruzi with the aim to elucidate the mechanism of action of these compounds on parasitic cells. Methods: The aims of the experiments were to detect and evaluate specifc events involved in apoptosis‑like cell death in the kinetoplastid, including DNA condensation, accumulation of reactive oxygen species and changes in ATP concentration, cell permeability and mitochondrial membrane potential, respectively, in treated cells. Results: The results demonstrated that the three isolated diterpenes could inhibit the tested parasites by inducing an apoptosis‑like cell death. Conclusions: These results encourage further investigation on the isolated compounds as potential drug candidates against both L. amazonensis and T. cruzi. Keywords: T. cruzi, L. amazonensis, Diterpenes, Marine compounds, Antikinetoplastid, Apoptosis‑like Background could represent a good candidate as a source of new com- Kinetoplastid parasites are responsible for a wide vari- pounds. Tese marine organisms are well known to pro- ety of infectious diseases, including Leishmania spp. and duce a wide variety of secondary metabolites and have Trypanosoma spp., which are causative agents of tropi- been investigated in several studies and shown to exhibit cal diseases classifed as neglected by the World Health various biological activities. Seaweeds have been source Organization. Tese diseases afect mainly countries material for the production of a variety of major metabo- with limited resources. Te appearance of resistance to lites that can be classifed in diferent groups according existing treatments as well as the side efects of these to their structure, such as polyphenols, polysaccharides, treatments motivate the search for new molecules as carotenoids, vitamins, lipids and terpenoid compounds, therapeutic alternatives [1, 2]. In this context, seaweeds among others [3]. Terpenes are secondary metabolites with two or more *Correspondence: [email protected] fve-carbon units of isopentenyl [4]. Tis class of mol- 1 Instituto Universitario de Enfermedades Tropicales y Salud Pública de ecules has been identifed as being biologically active, Canarias, Universidad de La Laguna (ULL), Avda. Astrofísico Fco. Sánchez with a proven efciency as anticancer, antioxidant, S/N, 38203 La Laguna, Tenerife, Spain Full list of author information is available at the end of the article anti-infammatory and antimicrobial properties. Tey © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Chiboub et al. Parasites Vectors (2021) 14:198 Page 2 of 13 are also known to represent the major class of metabo- Methods lites produced by marine algae [5]. Biological material and molecules identifcation Investigations on marine metabolites keep leading Te brown seaweed Dictyota spiralis was collected in the discovery of new structures and new activities. April 2017, and specimen identifcation was performed Over 170 diterpenoids, included in 32 patterns of car- by the National Institute for Marine Sciences and Tech- bon backbones, have been found to exhibit biological nologies in Tunisia. A crude extract of the collected activities, such as ichtyotoxicity, phytotoxicity, cyto- specimens was demonstrated to exhibit antikinetoplastid toxicity, antiviral, antifungal, insecticidal or antifeeding activity and was used to perform bio-guided fractiona- properties [6–8]. tion to isolate the active compounds. Several methods Te potential of these marine organisms to be a of separation and identifcation were used, including source of new biologically active molecules has inspired column chromatography, high-performance liquid chro- several investigations in drug discovery, including one matography, nuclear magnetic resonance, mass spec- on the screening of seaweed species collected from trometry, among others (Additional fle 1). Compound the Tunisian coast for antioxidant and antiprotozoal 1 was identifed as pachydictyol C [12], and compounds activities [9]. Te results of this investigation allowed 2 and 3 were characterized as dictyol E [12–14] and us to focus on the species Dictyota spiralis and its 3,4-epoxy-7,18-dolabelladiene [10], respectively. Stock crude organic extract that exhibited promising results solutions of these compounds were prepared in dimethyl in terms of antikinetoplastid capacity. A further inves- sulfoxide (DMSO) (Merck KGaA, Darmstadt, Germany), tigation employing bio-guided fractionation resulted protected from light, at − 20 °C. Te structure as well as in the isolation of specifc compounds possessing this the chemical names of the three diterpenes obtained are activity [10]. In this context, the research presented shown in Fig. 1. here focuses on the evaluation of the potential of these compounds as drug candidates. Terefore, the main Parasites strain, cytotoxicity and activity assays objective was to identify the mechanism of action of Experiments including in vitro activity assays of isolated these compounds on parasites at a molecular level and compounds 1–3 were performed against the promastig- to characterize the cell death pathway they induce as ote and amastigote stages of Leishmania amazonensis the latter is a determinant parameter in drug selection (MHOM/BR/77/LTB0016) and the epimastigote stage of as a prospective treatment. Cell death can be induced Trypanosoma cruzi (Y strain). Promastigotes of L. ama- in two main forms: regulated or accidental. Te frst zonensis were cultured in Schneider’s medium (Sigma- form is a modulated mechanism and the second form is Aldrich, St. Louis, MO, USA). Typanosoma cruzi strain instantaneous and catastrophic. Both pathways can be Y was cultured in liver Infusion tryptose (LIT) medium. identifed through the detection of macroscopic, mor- phological and molecular events [11]. In vitro antileishmanial evaluation Te aim of the present work was to detect the vari- Anti‑promastigotes assay ous events induced by exposing the kinetoplasts of cells Te bioassay was performed using the AlamarBlue® to three diferent metabolites isolated from the crude method as previously described [23]. Promastigotes extract of D. spiralis and to determine the cell death of L. amazonensis were grown at 26 °C in RPMI 1640 pathway induced by these molecules. modifed medium (Gibco BRL Life Technologies Inc., Gaithersburg, MD, USA) and supplemented with 10% Fig. 1 Chemical structure of three natural diterpenes isolated from Dictyota spiralis crude extract Chiboub et al. Parasites Vectors (2021) 14:198 Page 3 of 13 5 heat-inactivated fetal bovine serum (FBS). Cultures in the then counted, adjusted to 5 × 10 cells/ml, distributed logarithmic phase were seeded in sterilized 96-well micr- on the previous 96-well plate and incubated at 27 °C for otiter plates (Corning™, Corning, NY, USA) (10 6 para- 72 h. Te plate was observed under an inverted micro- sites/ml) containing the samples dissolved in 1% DMSO scope after 72 h of incubation and analyzed statistically at the suitable concentration to be tested in serial dilu- as described for the leishmanicidal test. tions, and Leishmania medium (RPMI 1640) was used to reach a fnal volume of 200 μl per well; then 10% of Assessment of cytotoxicity on macrophages AlamarBlue® was added to the entire plate. After an Cytotoxicity of the tested molecules was evaluated after incubation of 72 h, the plate was checked visually using 24 h using macrophages of the murine cell line J774. an inverted microscope, then analyzed using an Enspire A1 (American Type Culture Collection #TIB-67). Te multimode plate reader (PerkinElmer, Inc., Waltham, cells were maintained in DMEM medium at 37 ˚C in a MA, USA) at excitation and emission wavelengths of 570 5% CO2 humidifed incubator. Te viability of the

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