Avaliação de alguns aspectos da resposta imune tipo celular em animais portadores do tumor ascitico de Ehrlich e tratados com titanocenos

Orientador : Mary Luci de Souza QueirozTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias MedicasResumo: Neste trabalho, dando prosseguimento a estudos prévios realizados em nosso laboratório investigamos os efeitos do composto diciclopentadienildiclorotitânio IV, DDCT, (15 mg/kg/dia/ dois dias) sobre a capacidade funcional das células ¿natural killer¿ (NK) e a produção das citocinas do perfil Th1 [interleucina (IL)-2 e interferon-g (IFN-g)] e do perfil Th2 (IL-4 e IL-10) de animais normais e portadores do tumor ascítico de Ehrlich (TAE). Além disso, para efeito comparativo, acrescentamos ao nosso estudo o diciclopentadienilditiocianatotitânio IV, BCDT, o qual é um derivado do DDCT com substituições dos radicais halogênios do DDCT por pseudo-halogênios. Com o composto BCDT, além dos parâmetros imunológicos acima mencionados, avaliamos, os efeitos do tratamento com uma, duas e três doses de BCDT (10, 15 ou 30 mg/kg/dia) sobre o crescimento e diferenciação de precursores hematopoiéticos [Células formadoras de colônias de granulócitos/macrófagos (CFU-GM)], de animais normais e portadores do TAE, os quais foram previamente realizados com o DDCT. Nestes animais, avaliamos ainda a celularidade da medula óssea, a presença de fatores estimuladores de colônias hematopoéticas (CSFs ) e a sobrevida. Nossos resultados demonstraram uma maior eficácia do composto DDCT em relação ao BCDT. No entanto, apesar das diferenças encontradas, verificamos que estes titanocenos compartilham a habilidade de regular positivamente os desequilíbrios hematopoiéticos e imunológicos envolvidos na evolução temporal do TAE. Como já esperado, o TAE produziu, concomitante à mielossupressão, um aumento no número de CFU-GM esplênico e uma diminuição da celularidade da medula óssea. O tratamento dos animais portadores do TAE com o BCDT produziu, de forma dose-dependente, paralelamente a um aumento na sobrevida, um aumento da mielopoiese, uma redução no número de colônias esplênicas e uma restauração no número total e diferencial da medula óssea. A maior eficácia foi encontrada com a dose de 10 mg/kg/dia/ três dias, a qual, em animais normais, produziu melhor recuperação no número de CFU-GM na medula óssea e estimulou a produção de CSFs. Efeitos tóxicos foram observados nas maiores doses do composto associado à presença de hematopoiese esplênica e uma menor sobrevida dos animais. Quanto à produção das citocinas, o tratamento com os compostos impediu a polarização Th1-Th2 encontrada nos animais portadores do TAE. Neste sentido, o tratamento destes animais com o DDCT (15 mg/kg/dia/por dois dias) ou com o BCDT (10 mg/kg/dia/por 3dias), aumentou os níveis de secreção de IL-2, regulou positivamente a secreção atípica de IFN-g e reduziu os níveis de IL-10 aumentados durante a evolução temporal do TAE. O estudo da atividade funcional das células NK em animais portadores do TAE e tratados com os titanocenos demonstrou, especialmente com o DDCT, um aumento na capacidade citotóxica das células NK, a qual se encontrava reduzida. Estes resultados são encorajadores, uma vez que favorecem a utilização dos titanocenos em combinações terapêuticas com outros quimioterápicos visando reduzir a mielotoxicidade e suplementar a eficácia tumoricida destes agentesAbstract: In this work, as a continuation of previous studies in our laboratory, we investigated the effects of the titanocene dicyclopentadienyldichlorotitanium IV compound, DDCT, (15 mg/kg/day/ per two days) on the natural killer cell activity (NK) and on Th1 [interleukin (IL)-2 and interferon-g (IFN-g)] and Th2 (IL-4 and IL-10) responses in normal and Ehrlich ascites tumor (EAT)-bearing mice. Moreover, for comparative effect, we also studied the dicyclopentadienylditiocianatetitanium IV, BCDT. The structural difference between these compounds is the substitution of the halides halogens present in DDCT by pseudo halogens in BCDT. With the BCDT compound, besides the immunological parameters mentioned above, we evaluated the effect of the treatment using one, two or three doses of BCDT (10, 15 or 30 mg/kg/day) on the growth and differentiation of granulocyte-macrophage progenitor cells [colony-forming units of granulocyte-macrophage (CFU-GM)], of normal and EAT-bearing mice. As studied previously with the DDCT compound, we also evaluated the bone marrow cellularity, the presence of colony stimulating factors (CSFs) and survival of these animals. Our results demonstrated that the DDCT compound is more effective than the BCDT one. In despite of the differences found, we observed that these compounds share the ability of regulating positively the hematopoietic and immunologic unbalance during the EAT evolution. As expected for the EAT-model, concomitant myelosuppression, increased number of spleen CFU-GM and changes in bone marrow cellularity were observed. The treatment of EAT-bearing mice with BCDT produced a dose-dependent increase in myelopoiesis, a reduction in splenic colonies and a restoration of the total and differential marrow cell counts. In addition, BCDT treatments also increased the survival of these animals. The most effective dose schedule was 10 mg/kg/day/per three days, which, in normal animals, also produced increased bone marrow CFU-GM numbers along with a CSF production boost. High doses of BCDT cause toxic effects and induced extramedular hematopoiese. Regarding the production of cytokines, the treatment with the titanocene compounds blocked the Th1-Th2 polarization found during the EAT progression. In this respect, the treatment of these animals with DDCT (15 mg/kg/day/per two days) or BCDT (10 mg/kg/day/per three days) increased the IL-2 levels, regulated the atypical IFN-g secretion positively and reduced the levels of IL-10, increased during the temporal evolution of EAT. In relation to the functional activity of NK cells, the treatment with both compounds enhanced the NK cell function, reduced during the EAT growth. Only the DDCT compound rescued to normal values the activity of NK cells. These up-modulatory effects demonstrated in this study, specially using the DDCT compound, indicated a new aspect of the antitumoral action mechanism of the titanocenes. Thus, it is encouraging, in this context, to consider these compounds for combination chemotherapy, protecting the host from hematotoxicity as well as supplementing tumoricidal efficacyDoutoradoDoutor em Farmacologi

Synadenium umbellatum Pax. promotes cell cycle arrest and induces apoptosis in K-562 leukemia cells

A leucemia mielóide crônica (LMC) é uma doença mieloproliferativa clonal, que apresenta resistência à apoptose. A introdução do mesilato de imatinibe revolucionou o tratamento da LMC, porém a resistência ao imatinibe pode ser desenvolvida em qualquer tempo e, inevitavelmente, leva à progressão da doença. Synadenium umbellatum Pax. pertence à família Euphorbiaceae e é usado popularmente no Brasil para o tratamento do câncer. A citotoxicidade das Euphorbiaceae está associada com a capacidade dessas plantas e seus compostos bioativos em induzir apoptose em células tumorais. Portanto, este trabalho teve como objetivo investigar a citotoxicidade e os mecanismos de morte induzidos por S. umbellatum em células leucêmicas. A citotoxicidade de S. umbellatum foi avaliada pelo ensaio de exclusão do azul de tripano e a análise do ciclo celular foi feita por citometria de fluxo. Os mecanismos envolvidos na morte celular das células K-562 foram investigados por microscopia óptica e por citometria de fluxo. Os resultados demonstraram que S. umbellatum é citotóxico para células leucêmicas de uma maneira dependente da concentração. A análise morfológica revelou que o tratamento com S. umbellatum induziu as célula K-562 à morte por via apoptótica. Além disso, os dados indicam aumento de ERO S, alterações no potencial de membrana mitocondrial, externalização da fosfatidilserina e ativação de caspase 9. Em conjunto, os resultados demonstram que S. umbellatum promoveu retenção do ciclo celular das células K-562 e induziu estas células à morte por apoptose.Chronic myeloid leukemia (CML) is a clonal myeloproliferative disease that shows apoptosis resistance. The introduction of imatinib mesylate has revolutionized the treatment of CML, but imatinib resistance may develop at any time and inevitably leads to disease progression. Synadenium umbellatum Pax. belongs to the Euphorbiaceae family and is popularly used in Brazil for the treatment of cancer. The cytotoxicity of Euphorbiaceae is associated with the ability of these plants and their bioactive compounds to induce apoptotic tumor cell death. Therefore, we aimed to investigate the cytotoxicity and the mechanisms of death induced by S. umbellatum extract in leukemic cells. S. umbellatum cytotoxicity was evaluated by trypan blue exclusion assay and flow cytometric analysis of the cell cycle; the mechanisms involved in K-562 cell death were investigated by light microscopy and flow cytometry. The results demonstrate that S. umbellatum is cytotoxic to leukemic cells in a concentration-dependent manner. Morphological analysis revealed that S. umbellatum treatment induced K-562 cell death by an apoptotic pathway. Furthermore, data indicate ROS overproduction, alterations in mitochondrial membrane potential, phosphatidylserine externalization and activation of caspase 9. Taken together, the results demonstrate that S. umbellatum extract arrested the cell cycle and triggered apoptosis at several levels in K-562 cells

Avaliação de citotoxicidade de nanotransportadores sem substância ativa em função da sua densidade populacional

A par da evolução da nanotecnologia, assiste-se a uma crescente preocupação sobre eventuais riscos/toxicidade que os nanotransportadores possam representar [1]. A maioria dos trabalhos publicados relativos à avaliação da citotoxicidade in vitro de nanotransportadores (NT) incide na comparação quando a substância ativa (SA) é administrada em solução ou nanoencapsulada, sendo escassos os estudos que visam avaliar a possível toxicidade do NT por si só (sem SA). O objetivo deste trabalho consistiu na avaliação da citotoxicidade de NT sem SA em função da sua densidade populacional, tendo sido testados quatro NT distintos: nanocápsulas (NC), nanoesferas (NE), transportadores lipídicos nanoestruturados (NLC) e lipossomas, com oito densidades populacionais (diluições décimais de 2,10×1012 part/mL a 2,10×105 part/mL). A avaliação da citotoxicidade realizou-se numa cultura primária (linfócitos) pelo teste do MTT e numa linha celular (fibroblastos 3T3) pelo teste do vermelho neutro (NR)

Assessment of mutagenic and antimutagenic effects of Punica granatum in mice

In the present study, the ability of Punica granatum ethanolic leaf extract (PGL) and Punica granatum ethanolic fruit extract (PGF) to induce mutagenicity or to modulate the genotoxic effects induced by the alkylating agent cyclophosphamide (CP) was evaluated. Swiss male mice were treated by gavage for 10 days with PGL or PGF (12.5, 25, 50, and 75 mg/kg/day) prior to exposure to CP (i.p. 200 mg/kg), 24 h after the end of the treatment. Initial observations revealed that normal mice treated with both extracts (12.5, 25, 50, and 75 mg/kg/day) showed a similar micronucleated polychromatic erythrocyte (MNPCE) frequency to that of the control group. Investigation of the protective effect of PGL and PGF based on data analysis revealed that, irrespective of dose or extract, oral administration of PGL or PGF for 10 days prior to exposure had reduced, in a dose-dependent manner, the frequency of MNPCE induced by CP in all groups studied. Higher reductions were observed at PGF doses of 50 and 75 mg/kg. Taken together, these results demonstrate that mice treated with P. granatum showed an absence of mutagenic effects and dose-dependent protective effects against CP-induced oxidative DNA damage.No presente estudo investigamos o potencial do extrato etanólico das folhas da Punica granatum (PGFO) e do extrato etanólico dos frutos da Punica granatum (PGFR) de induzir mutagenicidade ou de proteger contra efeitos genotóxicos induzidos pela ciclofosfamida (CF). Camundongos machos Swiss foram tratados por 10 dias, via oral, com PGFO ou PGFR (12,5, 25, 50 e 75 mg/kg/dia), previamente a exposição à CF (i.p. 200 mg/kg) 24 horas após término do tratamento. Observamos que os animais tratados por 10 dias com ambos os extratos (12,5, 25, 50 e 75 mg/kg/dia) demonstraram a frequência de micronúcleo policromático eritrocitário (MNPCE) similar ao grupo controle. Quando aos efeitos protetores dos extratos foram investigados, a análise dos dados revelou que, independentemente da dose ou do extrato usado, a administração oral por 10 dias, previamente à exposição, reduziu, de forma dose-dependente, a frequência de MNPCE induzidos pela CF, em todos os grupos estudados. As maiores reduções foram observadas com PGFR nas doses de 50 e 75 mg/kg. Em conjunto, sob as condições testadas, camundongos tratados com P. granatum demonstraram ausência de efeitos mutagênicos e, de forma dose-dependente, efeitos protetores contra os danos oxidativos do DNA induzidos pela CF

Synadenium umbellatum: cytotoxicity and DNA damage to bone marrow cells from mice

In the present study we investigated, in vitro and in vivo, the cytotoxic and mutagenic potential of the Synadenium umbellatum ethanolic extract (SUEE) on the bone marrow cells of mice. In vitro cytotoxicity was assayed incubating bone marrow cells with different concentrations (40-0.312 mg/mL) of SUEE for 12, 24 or 48 h using the MTT tetrazolium reduction test and the trypan blue exclusion test. The micronuclei assay was performed to determine mutagenic effects of mice orally exposed to different doses of SUEE (10, 25 or 50 mg/kg/day) in 24 h. Our results demonstrated that SUEE has, in a dose-dependent manner, potential cytotoxic and mutagenic effects on the bone marrow cells of mice. Further studies are needed to expand the knowledge of the toxicological/pharmacological potential of the SUEE.No presente estudo investigamos o potencial citotóxico e mutagênico, in vitro e in vivo, respectivamente, do extrato etanólico de Synadenium umbellatum (EESU) sobre células da medula óssea de camundongos. A citotoxicidade in vitro foi avaliada por meio da exposição de células da medula óssea de animais normais a diferentes concentrações (40-0,312 mg/mL) do EESU, por 12, 24 ou 48 h, utilizando os testes de redução do MTT e o de exclusão do azul de tripano. O ensaio de micronúcleo foi realizado para investigar potenciais efeitos mutagênicos do EESU (10, 25 ou 50 mg/kg/dia) sobre a medula óssea de camundongos. Os animais foram expostos a uma única dose, por via oral, e 24 h após à exposição, sacrificados para realização do estudo (n=5/grupo). Os resultados obtidos demonstraram que o EESU possui potencial efeito citotóxico e mutagênico, de forma dose-dependente, sobre as células da medula óssea de camundongos, in vitro e in vivo, respectivamente. Maiores estudos são necessários para expandir o conhecimento acerca do potencial toxicológico/farmacológico do EESU

Biodegradable nanoparticles designed for drug delivery: The number of nanoparticles impacts on cytotoxicity

Nanostructured drug delivery systems are based on biocompatible and biodegradable components. Composition, size and membrane surface properties are characteristics that may influence cell viability in cytotoxicity assays. In this work, four nanostructured systems commonly used for drug delivery were prepared and cytotoxicity was evaluated on human lymphocytes and Balb/c 3T3 fibroblasts. The hemolytic potential was also investigated. Polymeric nanocapsules (NC) and nanospheres (NS), nanostructured lipid carriers (NLC) and liposomes were prepared and characterized for size, distribution, zeta potential and number per volume of the colloidal dispersion. Cell viability was evaluated, 24 and 48h, by MTT and neutral red assays (NR). Cells were incubated with each particle in eight different dilutions varying from 2.1×10 4 to 2.1×10 11 particles/mL. Diameter of nanoparticles was between 130 and 200nm, all samples exhibited narrow size distribution (polydispersity index below 0.1) and zeta potential varied from -6.8 to -19.5mV. NC, NS and NLC reduced cell viability in a dilution dependent manner. For these nanoparticles, the higher number of particles induced cell death for both cell types. Liposomes did not cause loss of cell viability even at the highest number of particles. Results suggest that, depending on the kind of nanoparticle, the number of particles in the dispersion can negatively influence cell viability in pre-clinical drug development.This work was supported by the Brazilian research funding agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de Estudos e Pesquisas (FINEP), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Apoio à Pesquisada Universidade Federal de Goiás (FUNAPE) and Fundação de Apoio à Pesquisa do Estado de Goiás (FAPEG).info:eu-repo/semantics/publishedVersio

Investigation of Ehrlich ascites tumor cell death mechanisms induced by Synadenium umbellatum Pax.

AbstractEthnopharmacological relevanceSynadenium umbellatum Pax. is widely found in South America and empirically used in Brazil for the treatment of several diseases, mainly cancer. The aim of the study was to investigate cell death mechanisms induced by Synadenium umbellatum Pax. using Ehrlich ascites tumor (EAT) cells, as well as the myelotoxicity potential of this plant.Materials and methodsS. umbellatum cytotoxicity was evaluated in EAT cells by trypan blue exclusion and MTT reduction test and the mechanisms involved in EAT cell death were investigated by light and fluorescence microscopy, flow cytometry and immunocytochemistry. Investigation of S. umbellatum myelotoxicity was performed by clonogenic assay of colony forming unit- granulocyte macrophage (CFU-GM).Results and ConclusionOur results demonstrated that S. umbellatum decreased the viability of EAT cells using both methods. Morphological analyses revealed that S. umbellatum-treatment induced EAT cell death by apoptotic pathway. We demonstrated the occurrence of reactive oxygen species (ROS) overgeneration, increased intracellular Ca2+ concentration, alteration in mitochondrial membrane potential, phosphatydylserine externalization, and activation of caspases 3, 8, and 9. However, S. umbellatum produced myelotoxicity in bone marrow cells in a concentration-dependent manner. In comparison to EAT cells, the effects of S. umbellatum in bone marrow cells were 8-fold lower. Taken together, our results showed that S. umbellatum induced apoptosis in EAT cells at several levels and seems more toxic to tumor cells than to normal bone marrow cells

Effects of a mucoadhesive formulation containing curcuma longa l. on oral wound healing

Introduction: The aim of this study was to investigate the effects of a topical mucoadhesive formulation with Curcuma longa L. extract (MFC) on oral wound healing. Methods: Seventy-two Wistar rats were randomly assigned to 3 groups: Control, Vehicle, and MFC. Traumatic ulcers were made on the dorsum of the tongue with a 3-mm diameter punch. Vehicle and MFC groups received application of the products twice a day, while animals in the control group were cared for in identical conditions but received no product application. Six rats in each group were euthanized at days 3, 5, 10, and 14. Percentage of repair was calculated based on wound area. HE-stained histological sections were obtained for semi-quantitative analysis of re-epithelization and inflammation. Results: Clinical findings revealed that at days 3 and 5, animals from the MFC group exhibited a significantly higher percentage of wound repair. At day 5, animals from this group also demonstrated a significant increase in the degree of re-epithelization and inflammation. Conclusions: MFC is capable of accelerating oral wound repair in an in vivo model by modulating the inflammatory process and stimulating epithelial proliferation

Grandisin induces apoptosis in leukemic K562 cells

In this study, the potential antileukemic activity of grandisin, a lignan extracted from Piper solmsianum, was evaluated against the leukemic line K562. The cytotoxicity of grandisin (0.018 to 2.365 µM) was evaluated in K562 and normal peripheral blood lymphocytes by Trypan Blue Exclusion and MTT methods after 48h exposure to the drug. In both methods, cellular viability was concentration-dependent and the IC50 values were lower than 0.85µM. Analysis of K562 cells after treatment with grandisin showed that the cell cycle was arrested in the G1 phase with a 12.31% increase, while both S and G2 phases decreased. Morphological studies conducted after the exposure of K562 to grandisin revealed changes consistent with the apoptosis process, which was confirmed by anexin V stain and caspase activation. Thus, lignan grandisin showed antileukemic activities against the K562 cell line and the cell death process occurred via apoptosis