Autophagy cell death process induced in Trypanosoma cruzi by piperovatine and piperlonguminine
Chagas disease is a neglected illness which currently affects 10–16 millions of people . As already demonstrated by our group, the amides piperovatine and piperlonguminine obtained from Piper ovatum Vahl have potent effect against epimastigote form of T. cruzi . In the present work, we report the activities of these compounds against proliferation of intracellular amastigote and also determine the mechanism of cell death in epimastigote using flow cytometry and labeling with monodansylcadaverine (MDC) techniques. Piperovatine and piperlonguminine concentration which inhibit 50% of growth (IC50) of amastigotes were 35.0±6.9µM and 33.9±5.4µM, respectively. Ultrastructural studies of the parasite showed alterations in cell membrane and presence of multiple vacuoles in the cytoplasm. Additionally, no alterations were observed in cell architecture of nucleus and mitochondria. For membrane integrity assay, treated parasites were incubated with 2mg/mL propidium iodide (PI) and analyzed in FACSCalibur flow cytometer (Becton-Dickinson, Rutherford, NJ, USA) equipped with the CellQuest software. This study showed an increase in PI signal, with 84% for piperovatine and 86% for piperlonguminine, suggesting alterations in membrane integrity. Besides that, in the study of cell death process, epimastigotes treated with these compounds were incubated with MDC (0.05 mM) and visualized using? uorescence microscope (Media cybernetics, US). Labeling with MDC revealed formation of autophagic vacuoles in 90% of the cells. These results showed in vitro that the mechanism of cell death can be by autophagic process, since the alterations induced had distinct feature from those of typical apoptosis or necrosis .
Acknowledgements: This study was supported through grants from CNPq, FINEP, Fundação Araucária, and CAPES.
References: 1. WHO. (2005) Report of the Scienti? c Working Group (SWG) on Chagas Disease, Buenos Aires, Argentina.
2. Veiga-Santos, P. et al. (2008) The biological activity of piperovatine and piperlonguminine isolated from Piper ovatum Vahl in epimastigote form of Trypanosoma cruzi. Planta Medica 75:877–1094.
3. Nguewa, P. A., et al. (2004) Programmed cell death in Trypanosomatids: a way to maximize their biological? tness? TRENDS in Parasitology 20:375–380.