RESUMO
Chagas' disease or American trypanosomiasis, caused by Trypanosoma cruzi infection, is an endemic disease in Latin America, which has spread worldwide in the past years. The drugs presently used for treatment have shown limited efficacy due to the appearance of resistant parasites and severe side effects. Some of the most recent studies on anti-parasitic drugs have been focused on protein acetylation, a reversible reaction modulated by Acetyl Transferases (KATs) and Deacetylases (KDACs). We have previously reported the anti-parasite activity of resveratrol (RSV), an activator of KDACs type III (or sirtuins), and showed that this drug can reduce the growth of T. cruzi epimastigotes and the infectivity of trypomastigotes. Since RSV is now widely used in humans due to its beneficial effects as an antioxidant, it has become an attractive candidate as a repurposing drug. In this context, the aim of the present study was to evaluate the ability of this drug to protect three different types of host cells from parasite infection. RSV treatment before parasite infection reduced the percentage of infected cells by 50-70% depending on the cell type. Although the mammalian cell lines tested showed different sensitivity to RSV, apoptosis was not significantly affected, showing that RSV was able to protect cells from infection without the activation of this process. Since autophagy has been described as a key process in parasite invasion, we also monitored this process on host cells pretreated with RSV. The results showed that, at the concentrations and incubation times tested, autophagy was not induced in any of the cell types evaluated. Our results show a partial protective effect of RSV in vitro, which justifies extending studies to an in vivo model to elucidate the mechanism by which this effect occurs.
Assuntos
Doença de Chagas , Parasitos , Trypanosoma cruzi , Humanos , Animais , Resveratrol/farmacologia , Resveratrol/uso terapêutico , Doença de Chagas/tratamento farmacológico , Doença de Chagas/prevenção & controle , Doença de Chagas/parasitologia , Acetilação , MamíferosRESUMO
TcTASV-C is a protein family of about 15 members that is expressed only in the trypomastigote stage of Trypanosoma cruzi. We have previously shown that TcTASV-C is located at the parasite surface and secreted to the medium. Here we report that the expression of different TcTASV-C genes occurs simultaneously at the trypomastigote stage and while some secreted and parasite-associated products are found in both fractions, others are different. Secreted TcTASV-C are mainly shedded through trypomastigote extracellular vesicles, of which they are an abundant constituent, despite its scarce expression on culture-derived trypomastigotes. In contrast, TcTASV-C is highly expressed in bloodstream trypomastigotes; its upregulation in bloodstream parasites was observed in different T. cruzi strains and was specific for TcTASV-C, suggesting that some host-molecules trigger TcTASV-C expression. TcTASV-C is also strongly secreted by bloodstream parasites. A DNA prime-protein boost immunization scheme with TcTASV-C was only partially effective to control the infection in mice challenged with a highly virulent T. cruzi strain. Vaccination triggered a strong humoral response that delayed the appearance of bloodstream trypomastigotes at the early phase of the infection. Linear epitopes recognized by vaccinated mice were mapped within the TcTASV-C family motif, suggesting that blockade of secreted TcTASV-C impacts on the settlement of infection. Furthermore, although experimental and naturally T. cruzi-infected hosts did not react with antigens from extracellular vesicles, vaccinated and challenged mice recognized not only TcTASV-C but also other vesicle-antigens. We hypothesize that TcTASV-C is involved in the establishment of the initial T. cruzi infection in the mammalian host. Altogether, these results point towards TcTASV-C as a novel secreted virulence factor of T. cruzi trypomastigotes.
Assuntos
Sangue/parasitologia , Doença de Chagas/parasitologia , Vesículas Extracelulares/parasitologia , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismo , Fatores de Virulência/metabolismo , Animais , Doença de Chagas/sangue , Doença de Chagas/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C3H , Família Multigênica , Transporte Proteico , Proteínas de Protozoários/genética , Trypanosoma cruzi/genética , Fatores de Virulência/genéticaRESUMO
Photodynamic therapy (PDT) is an anti-tumor treatment administered for the elimination of early-stage malignancies and the palliation of symptoms in patients with late-stage tumors, which involves the activation of a photosensitizer (PS) using light of a specific wavelength, which also generates singlet oxygen and other reactive oxygen species (ROS) that cause tumor cell death. Several mechanisms are involved in the protective responses to PDT including the expression of chaperone/heat shock proteins (HSPs). The HSPs are a family of proteins that are induced by cells in response to exposure to stressful conditions. In the last few decades, it has been discovered that HSPs can play an important role in cell survival, due to the fact that they are responsible for many cytoprotective mechanisms. These proteins have different functions depending on their intracellular or extracellular location. In general, intracellular HSPs have been related to an anti-apoptotic function and recently, HSP-induced autophagy has shown to have a protective role in these chaperones. In contrast, extracellular HSPs or membrane-bound HSPs mediate immunological functions. In the present article, we attempt to review the current knowledge concerning the role of HSPs in the outcome of PDT in relation to autophagy and apoptosis mediated-resistance to photodynamic treatment. We will also discuss how certain PDT protocols optimally stimulate the immune system through HSPs.
Assuntos
Morte Celular/imunologia , Proteínas de Choque Térmico/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fotoquimioterapia , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Humanos , Neoplasias/imunologiaRESUMO
Photodynamic therapy (PDT) employing methyl δ-aminolevulinic acid (Me-ALA), as a precursor of the photosensitizer protoporphyrin IX (PpIX), is used for the treatment of non melanoma cutaneous cancer (NMCC). However, one of the problems of PDT is the apparition of resistant cell populations. The aim of this study was to isolate and characterize squamous carcinoma cells SCC-13 resistant to PDT with Me-ALA. The SCC-13 parental population was submitted to successive cycles of Me-ALA-PDT and 10 resistant populations were finally obtained. In parental and resistant cells there were analyzed the cell morphology (toluidine blue), the intracellular PpIX content (flow cytometry) and its localization (fluorescence microscopy), the capacity of closing wounds (scratch wound assay), the expression of cell-cell adhesion proteins (E-cadherin and ß-catenin), cell-substrate adhesion proteins (ß1-integrin, vinculin and phospho-FAK), cytoskeleton proteins (α-tubulin and F-actin) and the inhibitor of apoptosis protein survivin, in the activated form as phospho-survivin (indirect immunofluorescence and Western blot). The results obtained indicate that resistant cells showed a more fibroblastic morphology, few differences in intracellular content of the photosensitizer, higher capacity of closing wounds, higher number of stress fibers, more expression of cell-substrate adhesion proteins and higher expression of phospho-survivin than parental cells. These distinctive features of the resistant cells can provide decisive information to enhance the efficacy of Me-ALA applications in clinic dermatology.