Estudo do mecanismo de ação do µ-diazidobis(N,N-dimetilbenzilamina)dipaládio(II) (CP2) sobre o metabolismo de Leishmania amazonensis (Kinetoplastida, Trypanosomatidae)
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Data
2018-10-31
Autores
Imamura, Kely Braga
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Universidade Estadual Paulista (Unesp)
Resumo
As leishmanioses são doenças mundialmente distribuídas, encontradas nas áreas tropicais e subtropicais do mundo e são causadas por protozoários parasitos do gênero Leishmania spp. A limitada disponibilidade de fármacos para o tratamento, somado à diversidade de efeitos colaterais e o surgimento de cepas resistentes, mostram a importância de pesquisas que sejam focadas no desenvolvimento de novas moléculas com alvos moleculares específicos e que contribuam para o tratamento destas, entre outras doenças negligenciadas. Como alternativa terapêutica, muitos complexos metálicos utilizados como quimioterápicos no tratamento do câncer têm sido testados em Tripanossomatídeos. Dentre estas moléculas, destaca-se o composto metálico CP2 (µ-diazidobis(N,N-dimetilbenzilamina)dipaládio(II)). Em estudos prévios, CP2 apresentou atividade leishmanicida e, em estudos in vivo, utilizando camundongos BALB/c, infectados com L. (L.) amazonensis, CP2 não apresentou toxicidade hepática ou renal e foi capaz de reduzir a carga parasitária dos animais de maneira similar à anfotericina B. CP2 demostrou, ainda, capacidade de inibir a etapa de clivagem da DNA Topoisomerase IB do parasito. Dessa forma, neste estudo, realizou-se a avaliação dos efeitos relacionados à pós-inibição da DNA TopoIB de Leishmania por CP2. Os resultados revelaram que CP2 induziu uma série de eventos celulares e moleculares em L. (L.) amazonensis. Promastigotas tratadas com CP2 em diferentes concentrações apresentaram a intensidade de marcação de Rho123 reduzida (apresentando valores negativos), nos tempos de 4 e 24h de tratamento (p <0,01), o que indica que o tratamento com CP2 induziu a despolarização da membrana mitocondrial. O nível de ROS aumentou nos parasitos dentro de 1h de tratamento após pré-incubação com o reagente H2DCFDA por 3h, e permaneceram aumentados ao longo de todo o período analisado (90 min). Estes resultados sugerem que há geração de ROS induzida por CP2. Quando as células de L. (L.) amazonensis foram tratadas com CP2 (4xIC50), Nac + CP2 (4xIC50), e Nac + CP2 (10xIC50), respectivamente, as células não conseguiram progredir para a fase M do ciclo celular, ficando paradas na fase S ao longo de todo o tempo analisado (8h). Interessantemente, as células tratadas somente com NAC progrediram naturalmente ao longo do ciclo, indicando que o efeito de parada no ciclo celular não é causado por ROS e sim, possivelmente, como consequência da inibição da DNATopoIB. A L. (L.) amazonensis respondeu a presença de HU, aumentando a fosforilação de H2A, em até 4,5 vezes. Entretanto, quando comparada à L. (L.) major percebe-se que a L. (L.) amazonensis apresenta níveis de fosforilação mesmo na ausência de estresse, indicando que naturalmente as células de L. (L.) amazonensis apresentam níveis basais de estresse celular. Quando as células foram expostas ao CP2, a porcentagem de células apoptóticas-like foi maior quando comparado às células necróticas em 6h de tratamento. Entretanto, o inverso foi obtido quando a porcentagem de células mortas foi observada após 48h de tratamento com CP2, indicando um aumento da população necrótica pós-apoptótica-like
Leishmaniasis are diseases globally distributed in tropical and subtropical areas of the world and Leishmania spp. are the etiological agents of the diseases. The limited availability of drugs for treatment, added to the diversity of collateral effects and the emergence of resistant strains, show the importance of research that is focused on the development of new molecules with specific molecular targets and that contribute to the treatment of these, among other diseases neglected. As a therapeutic alternative, many metal complexes used as chemotherapeutics in the treatment of cancer have been tested in trypanosomatids. Among these molecules, the metal compound CP2 (µ-diazidobis(N,N-dimetilbenzilamina)dipaládio(II)). In previous studies stands out. CP2 showed leishmanicidal activity and in in vivo studies using BALB/c mice infected with L. (L.) amazonensis, CP2 showed no hepatic or renal toxicity and was able to reduce the parasitic load of the animals in a manner similar to amphotericinB, CP2 has also been shown to inhibit the DNA cleavage stage Topoisomerase IB of the parasite. Thus, in this study, the effects related to post-inhibition of Leishmania TopoIB DNA by CP2 were evaluated. The results revealed that CP2 induced a series of cellular and molecular events in L. (L.) amazonensis. Promastigotes treated with CP2 in the different concentrations Showed Rho123 low intensity (exchange of negative values), at treatment times 4 and 24h (p <0.01), indicating CP2 treatment induced mitochondrial membrane depolarization. The level of ROS increased in the parasites within 1h of treatment after preincubation with the H2DCFDA reagent for 3h, and remained increased throughout the analyzed period (90 min). These results suggest that there is generation of ROS induced by CP2. When L. (L.) amazonensis cells were treated with CP2 (4xIC50), Nac + CP2 (10xIC50), and Nac + CP2 (10xIC50) respectively, the cells failed to progress to the M phase of the cell cycle, stops in the S phase throughout the analyzed time (8h). Interestingly, cells treated with NAC alone progressed naturally over the cycle, indicating that the stop effect on the cell cycle is not caused by ROS but possibly as a consequence of inhibition of DNATopoIB. The L. (L.) amazonensis responded to the presence of HU, increasing phosphorylation of H2A by up to 4.5 times. However, when compared to L. (L.) major it is noticed that L. (L.) amazonensis presents levels of phosphorylation even in the absence of stress, indicating that naturally L. (L.) amazonensis cells present basal levels of cellular stress. When cells were exposed to CP2, the percentage of apoptotic-like cells was higher when compared to necrotic cells in 6h of treatment. However, the inverse was obtained when the percentage of dead cells was observed after 48h CP2 treatment, indicating an increase in the necrotic post-apoptotic-like population.
Leishmaniasis are diseases globally distributed in tropical and subtropical areas of the world and Leishmania spp. are the etiological agents of the diseases. The limited availability of drugs for treatment, added to the diversity of collateral effects and the emergence of resistant strains, show the importance of research that is focused on the development of new molecules with specific molecular targets and that contribute to the treatment of these, among other diseases neglected. As a therapeutic alternative, many metal complexes used as chemotherapeutics in the treatment of cancer have been tested in trypanosomatids. Among these molecules, the metal compound CP2 (µ-diazidobis(N,N-dimetilbenzilamina)dipaládio(II)). In previous studies stands out. CP2 showed leishmanicidal activity and in in vivo studies using BALB/c mice infected with L. (L.) amazonensis, CP2 showed no hepatic or renal toxicity and was able to reduce the parasitic load of the animals in a manner similar to amphotericinB, CP2 has also been shown to inhibit the DNA cleavage stage Topoisomerase IB of the parasite. Thus, in this study, the effects related to post-inhibition of Leishmania TopoIB DNA by CP2 were evaluated. The results revealed that CP2 induced a series of cellular and molecular events in L. (L.) amazonensis. Promastigotes treated with CP2 in the different concentrations Showed Rho123 low intensity (exchange of negative values), at treatment times 4 and 24h (p <0.01), indicating CP2 treatment induced mitochondrial membrane depolarization. The level of ROS increased in the parasites within 1h of treatment after preincubation with the H2DCFDA reagent for 3h, and remained increased throughout the analyzed period (90 min). These results suggest that there is generation of ROS induced by CP2. When L. (L.) amazonensis cells were treated with CP2 (4xIC50), Nac + CP2 (10xIC50), and Nac + CP2 (10xIC50) respectively, the cells failed to progress to the M phase of the cell cycle, stops in the S phase throughout the analyzed time (8h). Interestingly, cells treated with NAC alone progressed naturally over the cycle, indicating that the stop effect on the cell cycle is not caused by ROS but possibly as a consequence of inhibition of DNATopoIB. The L. (L.) amazonensis responded to the presence of HU, increasing phosphorylation of H2A by up to 4.5 times. However, when compared to L. (L.) major it is noticed that L. (L.) amazonensis presents levels of phosphorylation even in the absence of stress, indicating that naturally L. (L.) amazonensis cells present basal levels of cellular stress. When cells were exposed to CP2, the percentage of apoptotic-like cells was higher when compared to necrotic cells in 6h of treatment. However, the inverse was obtained when the percentage of dead cells was observed after 48h CP2 treatment, indicating an increase in the necrotic post-apoptotic-like population.
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Palavras-chave
Compostos ciclopaladados, Instabilidade genômica, Ciclo Celular, Membrana mitocôndrial, Morte celular, Estresse oxidativo, Leishmania