Novas alternativas terapêuticas para o tratamento da Criptococose: análogos de Resveratrol e microRNAs
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Data
2016-02-29
Autores
Gullo, Fernanda Patrícia [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Criptococose é uma importante micose sistêmica que acomete principalmente pacientes imunocomprometidos e é classificada como a infecção fúngica com maior mortalidade entre indivíduos portadores de HIV. Cryptococcus neoformans é uma levedura capsulada e o principal agente etiológico da criptococose; encontra-se dispersa no meio ambiente na forma de basidiósporos, os quais são responsáveis pela infecção em humanos e animais. As principais manifestações clínicas estão associadas à infecção pulmonar e meningite. A terapia se dá basicamente com a administração de anfotericina B (AMB) na terapia de ataque associada ou não a 5-fluocitosina e como manutenção é indicado ao longo do tratamento o fluconazol (FCZ). Apesar de esta terapêutica ser eficiente, é constatado elevado número de casos de reincidência e desenvolvimento de resistência aos azóis, além de problemas de toxicidade. Diante desta problemática, este estudo propõe o desenvolvimento de novas alternativas terapêuticas para o tratamento da criptococose, visando duas abordagens distintas. A primeira, aplicando novo composto antifúngico e a segunda, o estudo de microRNAs (miRNAs) envolvidos na interação da levedura e células de glioblastoma humano (U87-MG), com a finalidade de usá-los como reguladores da infecção, sendo esta uma estratégia recentemente divulgada em uma variedade de doenças. Para tanto, inicialmente moléculas análogas de resveratrol foram avaliadas quanto a atividade antifúngica e o derivado orto,orto-HDZ (HDZ) mostrou forte atividade fungicida contra isolados de C. neoformans, com valores de concentração fungicida mínima (CFM) variando entre 0,97 a 7,81 µg/mL. A associação entre HDZ e FCZ mostrou sinergia com potencialização do efeito do FCZ em até 64 vezes para um isolado clínico resistente. Baixa toxicidade foi observada em ensaios in vitro e in vivo (Galleria mellonella e camundongos), apesar de mostrar alterações em embriões de Zebrafish. Além disso, este estudo mostrou a capacidade da associação HDZ + FCZ em inibir a interação da levedura e células hospedeiras (pneumócitos, macrófagos e glioblastoma) por citometria de fluxo e imunofluorescência. Ensaios in vivo (modelo murino) da atividade antifúngica de HDZ em combinação com FCZ mostrou que após 15 dias de tratamento houve redução significativa das unidades formadoras de colônias (UFC) no fígado, baço, cérebro, pulmões e lavado broncoalveolar, assim como o aumento da sobrevida dos animais em 198 dias e redução da morbidade, a qual foi avaliada por ensaio de comportamento (SHIRPA). Diante da segunda proposta deste trabalho, foram encontrados 10 miRNAs super-expressos na interação de C. neoformans e células U87-MG e foi observado que os miRNAs regulam principalmente vias de sinalização TGF-β, MAP quinase (MAPK) e interação receptor e matriz extracelular (MEC). Tais dados foram cruzados com transcritos que mostraram diferencialmente expressos na interação fungo-célula e as proteínas GTPase-3 e COP-1 foram selecionadas como importantes nesse processo. Tais proteínas estão associadas à via Rho-GTPase a qual é primordial para a ligação da levedura em células hospedeiras e mostrou aumento da expressão na infecção, assim como ligeira redução após o tratamento com HDZ. A indução da apoptose de células de glioblastoma por C. neoformans, foi observada através da técnica TUNEL e da expressão da proteína pró-apoptótica Bak e o tratamento com HDZ foi capaz de reverter esse processo. Nossos resultados indicam HDZ como uma interessante molécula para avançar nos estudos de desenvolvimento de um protótipo antifúngico contra C. neoformans e a inibição da expressão dos miRNAs durante a interação fungo-célula, pode ser interessante para controle da infecção fúngica, pois acreditamos que uma vez inibidos, podem reduzir a invasão da levedura em células hospedeiras, sendo então os miRNAs interessantes novos alvos terapêuticos e que o tratamento com HDZ são promissores para o desenvolvimento de um novo antifúngico.
Cryptococcosis is an important systemic mycosis that mainly affects immunocompromised patients and is classified as the fungal infection with higher mortality among individuals with HIV. Cryptococcus neoformans is an encapsulated yeast and the main etiologic agent of cryptococcosis. This yeast is dispersed into the environment in the form of basidiospores, which are responsible for infection in humans and animals. The main clinical manifestations are associated with pulmonary infection and meningitis. The treatment is basically the administration of amphotericin B (AMB) as therapy consolidation with or without the use of 5-flucytosine and, for the maintenance therapy, fluconazole (FCZ) is indicated. Although this therapy is effective, it is observed high number of cases of relapses, development of resistance to azoles and toxicity problems. In front of these problems, this study proposes the development of new therapies for the treatment of cryptococcosis, targeting two distinct approaches. The first aims a new antifungal compound and the second the study microRNAs (miRNAs) involved in the interaction between yeast and human glioblastoma cells (U87-MG), in order to use them as regulators of infection, since this is a recently disclosed strategy in a variety of diseases. For this purpose, initially, analogs of resveratrol molecule were evaluated for antifungal activity and the derived ortho,ortho-HDZ (HDZ) showed a strong fungicidal activity against isolates of C. neoformans with values of minimum fungicidal concentration (MFC) ranging between 0.97 to 7.81 µg/mL. The association between HDZ and FCZ showed synergy with potentiation of the effect of FCZ in up to 64 times for a resistant clinical isolate to FCZ. Low toxicity was observed in in vitro and in vivo assays (Galleria mellonella and mice), despite showing changes in Zebrafish embryos. In vivo assay (murine model) of the antifungal activity of HDZ in combination with FCZ showed that after 15 days of treatment, a significant reduction in colony forming units (CFU) in the liver, spleen, brain, lungs and bronchoalveolar lavage was observed, as well as, increased survival of the animals in 198 days with the reduction of the morbidity, which was evaluated by behavioral assay (SHIRPA). Considering the second purpose of this study, 10 miRNAs were over-expressed during the interaction of C. neoformans and U87-MG cells and it was noted that these miRNAs regulate the signaling pathway of TGF-β, MAP kinase (MAPK) and the receptor and matrix extracellular components (MEC) interaction. These data was cross with transcripts that showed differentially expressed in fungus-cell interaction and the GTPase-3 and COP-1 protein were selected as important in this process. Such proteins are associated to Rho-GT pathway Rho-GTPase which is essential for the yeast adhesion into host cells and showed increased expression in infection, as well as a slight reduction after treatment with HDZ. Induction of apoptosis in glioblastoma cells by C. neoformans was observed by TUNEL technique and the expression of pro-apoptotic protein Bak and treatment with HDZ was able to reverse this process. Our results indicate HDZ as an interesting molecule to advance the development of studies of a prototype antifungal against C. neoformans and that the inhibition of expression of miRNAs during fungus-cell interaction, may be interesting to control fungal infection, because we believe that once inhibited, the yeast can reduce invasion of host cells, being the miRNAs interesting new therapeutic targets and the treatment with HDZ is promising for the development of a new antifungal drug.
Cryptococcosis is an important systemic mycosis that mainly affects immunocompromised patients and is classified as the fungal infection with higher mortality among individuals with HIV. Cryptococcus neoformans is an encapsulated yeast and the main etiologic agent of cryptococcosis. This yeast is dispersed into the environment in the form of basidiospores, which are responsible for infection in humans and animals. The main clinical manifestations are associated with pulmonary infection and meningitis. The treatment is basically the administration of amphotericin B (AMB) as therapy consolidation with or without the use of 5-flucytosine and, for the maintenance therapy, fluconazole (FCZ) is indicated. Although this therapy is effective, it is observed high number of cases of relapses, development of resistance to azoles and toxicity problems. In front of these problems, this study proposes the development of new therapies for the treatment of cryptococcosis, targeting two distinct approaches. The first aims a new antifungal compound and the second the study microRNAs (miRNAs) involved in the interaction between yeast and human glioblastoma cells (U87-MG), in order to use them as regulators of infection, since this is a recently disclosed strategy in a variety of diseases. For this purpose, initially, analogs of resveratrol molecule were evaluated for antifungal activity and the derived ortho,ortho-HDZ (HDZ) showed a strong fungicidal activity against isolates of C. neoformans with values of minimum fungicidal concentration (MFC) ranging between 0.97 to 7.81 µg/mL. The association between HDZ and FCZ showed synergy with potentiation of the effect of FCZ in up to 64 times for a resistant clinical isolate to FCZ. Low toxicity was observed in in vitro and in vivo assays (Galleria mellonella and mice), despite showing changes in Zebrafish embryos. In vivo assay (murine model) of the antifungal activity of HDZ in combination with FCZ showed that after 15 days of treatment, a significant reduction in colony forming units (CFU) in the liver, spleen, brain, lungs and bronchoalveolar lavage was observed, as well as, increased survival of the animals in 198 days with the reduction of the morbidity, which was evaluated by behavioral assay (SHIRPA). Considering the second purpose of this study, 10 miRNAs were over-expressed during the interaction of C. neoformans and U87-MG cells and it was noted that these miRNAs regulate the signaling pathway of TGF-β, MAP kinase (MAPK) and the receptor and matrix extracellular components (MEC) interaction. These data was cross with transcripts that showed differentially expressed in fungus-cell interaction and the GTPase-3 and COP-1 protein were selected as important in this process. Such proteins are associated to Rho-GT pathway Rho-GTPase which is essential for the yeast adhesion into host cells and showed increased expression in infection, as well as a slight reduction after treatment with HDZ. Induction of apoptosis in glioblastoma cells by C. neoformans was observed by TUNEL technique and the expression of pro-apoptotic protein Bak and treatment with HDZ was able to reverse this process. Our results indicate HDZ as an interesting molecule to advance the development of studies of a prototype antifungal against C. neoformans and that the inhibition of expression of miRNAs during fungus-cell interaction, may be interesting to control fungal infection, because we believe that once inhibited, the yeast can reduce invasion of host cells, being the miRNAs interesting new therapeutic targets and the treatment with HDZ is promising for the development of a new antifungal drug.
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Palavras-chave
Cryptococcus neoformans, Resveratrol, MicroRNA