This study presents morphological and biochemical evidence of programmed cell death (PCD) in Entamoeba histolytica induced by exposure of trophozoites to the aminoglycoside antibiotic G418. Morphological characteristics of PCD, including cell shrinkage, reduced cellular volume, nuclear condensation, DNA fragmentation and vacuolization were observed, with preservation of trophozoite membrane integrity. PCD is orchestrated biochemically by alterations in intracellular ion fluxes. In G418-treated trophozoites, overproduction of reactive oxygen species (ROS), decreased intracellular K + , increased cytosolic calcium, and decreased intracellular pH levels were observed. However, externalization of phosphatidylserine was not detected. These results suggest that amoebae can undergo PCD under stress conditions, and that this PCD shares several properties with PCD reported in mammals and in a variety of unicellular organisms. INTRODUCTIONEntamoeba histolytica, the causal agent of amoebiasis, is a protozoan parasite that resides in the colon of infected humans. The invasive trophozoites adhere to mucus and epithelial cells, proliferate by binary fusion, and release proteolytic factors that destroy the intestinal mucosa, resulting in amoebic dysentery. In one in 10 patients with intestinal E. histolytica infection, the trophozoites migrate through the portal vein to the liver and give rise to amoebic abscesses, the main cause of death by this parasite (Espinosa-Cantellano & Martínez-Palomo, 2000). The apoptosis of host cells such as macrophages induced by contact with E. histolytica trophozoites has been widely studied, and it is considered an important feature of the host-parasite relationship (Ragland et al., 1994;Berninghausen & Leippe, 1997).Programmed cell death (PCD) has been considered a critical mechanism of development, differentiation and control of cellular proliferation in metazoans. However, increasing evidence indicates that PCD is also present in unicellular organisms. Forms of PCD such as apoptosis, apoptosis-like processes and necrosis-like processes have been identified in several bacteria (Lewis, 2000), yeast (Madeo et al., 1999), the slime mould Dictyostelium discoideum (Cornillon et al., 1994), the dinoflagellate Peridinium gatunense (Vardi et al., 1999), the euglenoid Euglena gracilis (Scheuerlein et al., 1995), the ciliate Tetrahymena thermophila (Christensen et al., 1995), and the protozoan parasites Trypanosoma, Leishmania (Nguewa et al., 2004) and Plasmodium (Al-Olayan et al., 2002). Recently, results reported by Ramos et al. (2007) have suggested the induction of an apoptotic-like process by nitric oxide species in E. histolytica. Apoptosis is the result of a genetic program that induces cellular and biochemical changes, including caspase activation, externalization of phosphatidylserine (PS), an increase in intracellular Ca 2+ and mitochondrial dysfunction, as well as physical changes such as cell shrinkage, alteration in cell volume, cytoplasmic blebbing and vacuolization, chromatin conden...
BackgroundDecatropis bicolor (Zucc.)Radlk is a plant that has been traditionally used for the treatment of breast cancer in some communities of Mexico. So, the aim of this study was to determine the cytotoxic and apoptotic effect of the essential oil of Decatropis bicolor against breast cancer cell line, MDA-MB-231.MethodsThe essential oil obtained from hydrodestillation of leaves of Decatropis bicolor was studied for its biological activity against breast cancer cells MDA-MB-231 by MTT assay, Hematoxylin-eosin stain, Annexin V-FITC, TUNEL and western blot assays and for its chemical composition by GC-MS.ResultsThe results showed a relevant cytotoxic effect of the essential oil towards MDA-MB-231 cells in a dose- and time- dependent manner, with an IC50 of 53.81 ± 1.691 μg/ml but not in the epithelial mammary cell line MCF10A (207.51 ± 3.26 μg/ml). Morphological examination displayed apoptotic characteristics in the treated cells like cell size reduction, membrane blebbing and apoptotic bodies. In addition, the apoptotic rate significantly increased as well as DNA fragmentation and western blot analysis revealed that the essential oil induced apoptosis in the MDA-MB-231 cells via intrinsic pathways due to the activation of Bax, caspases 9 and 3. Phytochemical analysis of the Decatropis bicolor essential oil showed the presence of twenty-three compounds. Major components of the oil were 1,5-cyclooctadiene,3-(methyl-2)propenyl (18.38 %), β-terpineol (8.16 %) and 1-(3-methyl-cyclopent-2-enyl)-cyclohexene (6.12 %).ConclusionsThis study suggests that essential oil of Decatropis bicolor has a potential cytotoxic and antitumoral effect against breast cancer cells, with the presence of potential bioactive compounds. Our results contribute to the validation of the anticancer activity of the plant in Mexican traditional medicine.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-016-1136-7) contains supplementary material, which is available to authorized users.
Oxygen or nitrogen oxidative species and chemical stress induce the programmed cell death (PCD) of Entamoeba histolytica trophozoites. PCD caused by the aminoglycoside G418 is reduced by incubation with the cysteine protease inhibitor E-64; however, no typical caspases or metacaspases have been detected in this parasite. Calpain, a cysteine protease activated by calcium, has been suggested to be part of a specific PCD pathway in this parasite because the specific calpain inhibitor Z-Leu-Leu-Leu-al diminishes the PCD of trophozoites. Here, we predicted the hypothetical 3D structure of a calpain-like protein of E. histolytica and produced specific antibodies against it. We detected the protein in the cytoplasm and near the nucleus. Its expression gradually increased during incubation with G418, with the highest level after 9 h of treatment. In addition, a specific calpain-like siRNA sequence reduced the cell death rate by 65%. All these results support the hypothesis that the calpain-like protein is one of the proteases involved in the execution phase of PCD in E. histolytica. The hypothetical interactome of the calpain-like protein suggests that it may activate or regulate other proteins that probably participate in PCD, including those with EF-hand domains or other calcium-binding sites.
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