PINHEIRO, D. P. Avaliação do potencial citotóxico e mecanismo molecular das naftoquinonas sintéticas ENSJ39 e ENSJ108: estudos in vitro e in silico. 2017. 213 f. Tese ( Doutorado em Farmacologia) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2017. / Submitted by Farmacologia Pós-Graduação (posgfarmacologia@gmail.com) on 2017-10-26T16:51:14Z No. of bitstreams: 1 2017_tese_dppinheiro.pdf: 333951 bytes, checksum: 50509ba3b73b6c364f0d8ad0bff04928 (MD5) / Approved for entry into archive by Maria Carvalho (lolitaprado@ufc.br) on 2017-10-30T11:26:13Z (GMT) No. of bitstreams: 1 2017_tese_dppinheiro.pdf: 333951 bytes, checksum: 50509ba3b73b6c364f0d8ad0bff04928 (MD5) / Made available in DSpace on 2017-10-30T11:26:13Z (GMT). No. of bitstreams: 1 2017_tese_dppinheiro.pdf: 333951 bytes, checksum: 50509ba3b73b6c364f0d8ad0bff04928 (MD5) Previous issue date: 2017-07-31 / Cancer consists in a set of diseases, which present in common the disordered proliferation and the inability of normal cellular differentiation. It is a disease of high incidence and one of the main causes of mortality worldwide, with an estimate for Brazil, in the biennium 2016-2017, indicating the occurrence of about 600 thousand new cases. Despite the large arsenal of chemotherapy in clinic, several studies are conducted in the search for drugs with higher therapeutic potency and, especially, more selective for tumor cells. Naphthoquinones are object of several studies due to their pharmacological activities, presenting excellent antitumor activity, besides microbicidal and anti-inflammatory activities, appearing as good candidates for the cancer treatment. Thus, the present study aimed to evaluate the in vitro cytotoxic effect of naphthoquinone compounds, as well as to study the mechanisms of action involved in the process of cell death induction by these compounds. Therefore, the compounds were initially subjected to a target-directed screening as inhibitors of topoisomerases and DNA repair enzymes, as well as evaluation of cell viability in several tumor and non-tumor cell lines. From the initial group of compounds, two were selected for further evaluation of the mechanisms of action: synthetic naphthoquinone ENSJ39, analogous to Nor-β-lapachone; and ENSJ108, analogous to α-lapachone. The results showed that, in the HCT-116 cancer cell line, the naphthoquinone ENSJ39, bioactivated by the NQO1 enzyme, induced an antiproliferative effect followed by a cytotoxic effect in a concentration-dependent manner, inhibiting cell cycle progression with G2/M arrest. These effects are related to the DNA damage caused by the reactive oxygen species generated by the drug. DNA damage activates the NHEJ and HR repair pathways, and is also related to the drug-induced topoisomerase II inhibition, which leads to the trapping of Top2cc; besides of a possible catalytic inhibition of topoisomerase I. These effects may induce death by apoptosis via the mitochondrial (intrinsic) pathway or, secondarily, appear to activate the extrinsic pathway. The naftoquinone ENSJ108, in tumor cells HCT-116, induced an antiproliferative effect followed by a cytotoxic effect, in a concentration-dependent manner, promoting the generation of reactive oxygen species (ROS). It results in a ROS-dependent DNA damage, leading to hyperactivation of PARP1, culminating in a type of cell death due to regulated necrosis called parthanatos. The drug also causes topoisomerase II inhibition by top2cc trapping, resulting in double-strand breaks and activation of TDP2 and HR. Secondarily, the drug appears to activate apoptosis via the extrinsic pathway. In conclusion, the naphthoquinones ENSJ39 and ENSJ108 show potent in vitro antitumor activity, highlighting the importance of future studies in animal models to evaluate the therapeutic potential of these molecules. / O câncer compreende um conjunto de doenças, que apresentam em comum a proliferação desordenada e a incapacidade de diferenciação celular normal. É uma doença de alta incidência e uma das principais causas de mortalidade em todo mundo, com estimativa para o Brasil, no biênio 2016-2017, apontando a ocorrência de cerca de 600 mil novos casos. Apesar do grande arsenal existente de quimioterápicos na clínica, vários estudos são realizados na busca por fármacos com maior potência terapêutica e, especialmente, mais seletivos para as células tumorais. As naftoquinonas são objeto de diversos estudos devido suas atividades farmacológicas, apresentando excelente atividade antitumoral, além de atividades antimicrobiana e anti-inflamatória, surgindo como bons candidatos para o tratamento do câncer. Dessa forma, o presente estudo teve por finalidade avaliar o efeito citotóxico in vitro de compostos naftoquinonas, bem como, estudar os mecanismos de ação envolvidos no processo de indução de morte celular destes compostos. Os compostos pertencentes ao grupo das naftoquinonas sintéticas foram inicialmente submetidos a um screening alvo-dirigido como inibidores de topoisomerases e enzimas de reparo de DNA, além de avaliação da viabilidade celular em diversas linhagens celulares tumorais e não tumorais. Do grupo inicial de compostos, dois foram selecionados para prosseguimento da avaliação dos mecanismos de ação: a naftoquinona sintética ENSJ39, análogo da Nor-β-lapachona; e a ENSJ108, análogo da α-lapachona. Os resultados mostraram que, na linhagem tumoral HCT-116, a naftoquinona ENSJ39, bioativada pela enzima NQO1, induziu efeito antiproliferativo seguido de efeito citotóxico, de maneira concentração dependente, inibindo a progressão do ciclo celular com parada em G2/M. Esses efeitos estão relacionados ao dano de DNA causado pelas espécies reativas de oxigênio geradas pela droga. O dano ao DNA ativa as vias de reparo por NHEJ e HR, estando também relacionado à inibição de topoisomerase II induzida pela droga, que leva ao aprisionamento de Top2cc; além de uma possível inibição catalítica da topoisomerase I. Estes efeitos podem induzir a morte por apoptose pela via mitocondrial (intrínseca) ou, de forma secundária, parece ativar a via extrínseca. Já a naftoquinona ENSJ108, em células tumorais HCT-116, induziu efeito antiproliferativo seguido de efeito citotóxico, de maneira concentração dependente, promovendo geração de espécies reativas de oxigênio (ROS). As ROS geradas causam dano ao DNA, levando a hiperativação de PARP1 que culmina com um tipo de morte celular por necrose regulada chamada parthanatos. A droga causou também inibição de topoisomerase II, pelo aprisionamento de topo2cc, resultando em quebras de dupla fita e ativação de TDP2 e HR. De forma secundária, a droga parece ativar apoptose pela via extrínseca. Podemos concluir que as naftoquinonas ENSJ39 e ENSJ108 mostraram potente atividade antitumoral in vitro, destacando a importância de futuros estudos em modelos animais para avaliação do potencial terapêutico destas moléculas.

The degree of DNA supercoiling in the cell is carefully controlled by DNA topoisomerases. These enzymes catalyze the passage of individual DNA strands (Type I DNA topoisomerases), or double helices (Type II DNA topoisomerases) through one another. The purpose of the present study is to conduct a detailed analysis of the topo llα and β mRNAs expressed in several vertebrate cell lines. The final aim of this project is to analyze the relative roles of topo llα in chromatin condensation and chromosome segregation during mitosis, by performing topo llα gene targeting experiments in the DT 40 chicken lymphoblastoid cell line. The knock-out strategy was based on the observation of a high rate of homologous recombination versus random integration in the DT40 cell line. The topo llα gene was shown to be located on the chicken chromosome 2 (APM unpublished), for which the DT40 cell line is trisomic. The targeting vector completely replaced the 32 kb topo IIα genomic locus, generating a topo llα (-/+/+)cell line, which showed an increased resistance to topo II inhibitors. Paradoxically, 150 uM etoposide or 100 uM mitoxanthrone induced apoptosis within 5 hours in the topo llα (-1+1+) cell line, more rapidly as compared to the normal DT 40 cells. A topo IIα (-I-I+) cell line has also been generated. This study revealed the presence of evolutionarily conserved alternatively spliced forms of both topo llα and β isoforms between birds and humans. Hybridization screening of two chicken cDNA libraries, MSB-1 and DU249, revealed the presence of two distinct forms of both topo llα and β cDNAs. One form of topo llα, designated topo llα-1, encodes the chicken topo llα amino acid sequence previously reported (dbjiAB007445) in the database (unpublished). The second form, designated topo llα-2, encodes a protein containing an additional 35 amino acids inserted after Lysine-322 of chicken topo IIα-1 protein sequence. In the case of topo 11(3 mANA, one form, designated topo IIβ-1, encodes the protein already described (dbjiAB007446). The second form, tapa IIβ-2, would encode a protein missing the next 86 amino acids after Valine-25 in tapa II β-1 protein sequence. The tapa 11β variant is positioned similarly to one previously described in human (Hela) cells. The 5 amino acid insertion in the human tapa 11β-2 variant follows v23. In chicken cells, a longer insertion of 86 amino acids sequence follows v25, the homologous position in the tapa 11β protein. In human cells, the situation with tapa llα is more complex, as revealed by RT-PCR experiments (APM, unpublished) which generated several bands. One of these amplified species was found to contain a 36 amino acids insertion, positioned after residue K321 in the human tapa IIα cDNA, similarly to chicken tapa IIα-2 variant. The second human tapa llα spliced form cDNA was shown to contain a 26 amino acids insertion after residue A401 in the canonical human tapa llα protein sequence. The third cDNA variant isolated from human cells was described to encode a 81 amino acids insertion after residue Q355 positioned within the known human tapa IIα protein. It appears possible that the posttranslational modifications of the a-2 and β-2 isoforms may differ substantially from those of the canonical a-1 and β-1 isoforms. Such variant proteins could fulfil specialized functions, which might be tissue or cell-type specific. In summary, two novel forms of tapa llα and β cDNAs have been identified in three chicken cell lines. These spliced versions of both tapa llα and 13 isoforms seem to be evolutionary conserved, with similar forms occurring in their human counterparts. Future functional analysis of vertebrate tapa IIα and β will have to account for the existence of these novel isoforms, which might encode proteins that may exhibit different regulation of their subcellular localization, interaction with other proteins, or catalytic activity.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Thesis (M.S.)--Rutgers University, 2009. / "Graduate Program in Microbiology and Molecular Genetics." Includes bibliographical references (p. 49-53).

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Thesis (Ph.D)--University of Tennessee Health Science Center, 2008. / Title from title page screen (viewed on July 16, 2007). Research advisor: Mary-Ann Bjornsti, Ph.D. Document formatted into pages (xii, 123 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 118-123).

Thesis (Ph.D.)--University of Tennessee Health Science Center, 2007. / Title from title page screen (viewed on July 29, 2008). Research advisor: Mary-Ann Bjornsti, Ph.D. Document formatted into pages (xiii, 175 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 161-175).

SOARES, Bruno Marques. Hellebrigenina, um bufodienolídeo com potencial ação compatível de inibidor catalítico da Topoisomerase II. 2013. 85 f. Dissertação (Mestrado em Farmacologia) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2013. / Submitted by denise santos (denise.santos@ufc.br) on 2013-08-28T15:47:03Z No. of bitstreams: 1 2013_dis_bmsoares.pdf: 2334333 bytes, checksum: 332b2a9235ebad6d7da12ab6ac20ee16 (MD5) / Approved for entry into archive by denise santos(denise.santos@ufc.br) on 2013-08-28T15:49:37Z (GMT) No. of bitstreams: 1 2013_dis_bmsoares.pdf: 2334333 bytes, checksum: 332b2a9235ebad6d7da12ab6ac20ee16 (MD5) / Made available in DSpace on 2013-08-28T15:49:38Z (GMT). No. of bitstreams: 1 2013_dis_bmsoares.pdf: 2334333 bytes, checksum: 332b2a9235ebad6d7da12ab6ac20ee16 (MD5) Previous issue date: 2013 / Bufodienolides are cardioactive steroids of 24 carbons, originally isolated from a frog’s skin extract of the family Bufonidae used in Chinese medicine. Bufodienolides shows many biological activities, including anticancer activities. Related to antitumor activity, the bufodienolídeos has been shown to inhibit the growth of several human cancer cell lines by inducing apoptosis and cell cycle arrest. This study evaluated the potential cytotoxicity and genotoxicity of six bufodienolides, in six human tumor cell lines, three normal murine lineages and PBMC (peripheral blood mononuclear cells). All six bufodienolides were cytotoxic to all cell lines and tumor PBMC with IC50 values ranging from 0.002 to 3.17 µM. Bufodienolides showed no cytotoxicity for normal murine strains. Thus, the compound hellebrigenin was chosen to determine the action mechanism involved, a sequence of in vitro experiments were performed using HL-60 leukemia cell line. Cells were treated at different concentrations of hellebrigenin (0.03, 0.06 and 0.12 µM) for 24 hours. Cell viability (viable cell number and membrane integrity) HL-60 assessed by flow cytometry showed that the number of cells decreased from the lower concentration (0.03 µM) tested and the percentage of cells with reduced membrane integrity from 0.06 µM concentration. Morphological analysis by flow cytometry revealed increased apoptotic cells starting at concentrations of 0.06 µM. The analysis of nuclear content, showed an increase in DNA fragmentation indicative of sub-G1 apoptosis and accumulation of cells in G2 / M phase from the concentrations of 0.03 and 0.06 µM, respectively. Other tests by flow cytometry revealed that there was an externalization of phosphatidylserine, mitochondrial depolarization, activation of caspase 8 and initiating subsequent activation of effector caspases 3 and 7. These data indicate a cytotoxic mechanism induced by over an apoptotic pathway. Hellebrigenin was not able to cause DNA damage in HL-60 and PBMC nor the emergence of chromosomal aberrations in PBMC. Through the studies of molecular docking was possible to predict the connection between hellebrigenina and human topoisomeraseIIα, showing a result that is compatible with a possible inhibition of this enzyme. Overall, the results indicate the potential cytotoxicity of hellebrigenin. / Os bufodienolídeos são esteróides cardioativos de 24 carbonos, isolados originalmente de um extrato de pele de sapos da família Bufonidae utilizado na medicina chinesa. Os bufodienolídeos possuem grande variedade de atividades biológicas, incluindo atividades antineoplásicas. Em relação à atividade antitumoral, os bufodienolídeos tem demonstrado inibir o crescimento de várias linhagens de células cancerígenas humanas por induzir apoptose e parada do ciclo celular. O presente estudo avaliou o potencial citotóxico e genetóxico de seis bufodienolídeos em seis linhagens tumorais humanos, três linhagens murinas normais e células mononucleadas do sangue periférico (CMSP) humano. Todos os seis bufodienolídeos foram citotóxicos para todas as linhagens tumorais e CMSP com valores de IC50 variando entre 0,002 e 3,17 µM. Os bufodienolídeos testados não apresentaram citotoxicidade para linhagens murinas normais. Desta forma, o composto hellebrigenina foi escolhido para se determinar o mecanismo de ação envolvido. Uma sequência de experimentos in vitro foram realizados utilizando-se a linhagem leucêmica HL-60. As células foram tratadas em diferentes concentrações da amostra hellebrigenina (0,03, 0,06 e 0,12 µM) por 24 horas. A viabilidade das células (número de células viáveis e integridade de membrana) HL-60 avaliada por citometria de fluxo, mostrou que o número de células reduziu a partir da menor concentração (0,03 µM) testada e a porcentagem de células com membrana integra reduziu a partir da concentração 0,06 µM. A análise morfológica por citometria de fluxo revelou aumento de células com padrão apoptótico a partir da concentração de 0,06 µM. Já a análise do conteúdo nuclear, nos mostrou aumento de fragmentação de DNA sub-G1 indicativo de apoptose e acúmulo de células na fase G2/M a partir das concentrações de 0,03 e 0,06 µM, respectivamente. Outros testes por citometria de fluxo revelaram que houve externalização da fosfatidilserina, despolarização mitocondrial, ativação da caspase iniciadora 8 e consequente ativação das caspases efetoras 3 e 7. Estes dados indicam um mecanismo citotóxico por indução de mais de uma via apoptótica. Hellebrigenina não foi capaz de causar danos ao DNA de HL-60 e de CMSP e nem o surgimento de aberrações cromossômicas em CMSP. Por meio dos estudos de docking molecular foi possível predizer a ligação entre hellebrigenina e topoisomeraseIIα humana, resultado compatível com a possível inibição dessa enzima. De forma geral, os resultados apontam o potencial citotóxico do bufodienolídeo hellebrigenina.

Made available in DSpace on 2015-07-22T20:50:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-03-26. Added 1 bitstream(s) on 2015-08-11T03:25:45Z : No. of bitstreams: 1 Publico-10807.pdf: 786945 bytes, checksum: a640250d88b5bd045dc6f2f53834bd45 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Objetivos: Estudar a expressao imuno-histoquimica de topoisomerase IIƒ¿ e de caspase-3 ativada, marcadores de proliferacao e de apoptose, respectivamente, a deteccao de DNA HPV e a evolucao da lesao cervical em mulheres portadoras de lesao intra-epitelial escamosa de baixo grau (LBG). Metodos: Foram avaliadas 40 mulheres portadoras de LBG e 32 sem neoplasia cervical, diagnosticadas por exame cito-colpo-histopatologico, quanto a imunoexpressao de topoisomerase IIƒ¿ e de caspase-3 ativada e quanto a deteccao de DNA HPV por PCR consensual (GP5+/GP6+) em material de esfregaco cervico-vaginal. Os achados foram relacionados as variaveis clinicas das pacientes e a evolucao clinica das lesoes cervicais em 12 meses. As pacientes assinaram termo de consentimento livre e esclarecido. Resultados: A media percentual de celulas imunomarcadas por topoisomerase foi de 11,71% e 4,13%, no grupo com LBG e controle, respectivamente, com diferenca estatisticamente significante. Observou-se que houve expressao de caspase-3 em 17 (42,5%) e em 5 (15,63%) pacientes com e sem LBG, respectivamente, com diferenca estatisticamente significante. Foi detectado HPV DNA em 65% das pacientes com LBG e em 59,4% das pacientes sem lesao cervical, sem relacao com a expressao de topoisomerase IIƒ¿ ou caspase-3. Na presenca de DNA-HPV, a expressao de topoisomerase IIƒ¿ no grupo com LBG foi significativamente maior do que em fragmentos sem lesao. Nao foi observada diferenca quanto a evolucao da lesao cervical em 12 meses de acordo com a imunoexpressao de topoisomerase IIƒ¿. Com relacao a caspase-3 ativada, a maioria das pacientes com imuno-histoquimica negativa teve regressao da lesao cervical. Conclusoes: A imunoexpressao de topoisomerase IIƒ¿ e de caspase-3 ativada podem ser considerados marcadores de proliferacao e de apoptose em lesao cervical de baixo grau, sem relacao com a presenca de DNA-HPV. / Purpose: To evaluate the correlation between the expression of topoisomerase II alpha, active caspase-3 and infection with human papillomavirus in low-grade cervical intraepithelial lesion and in the normal cervix, and whether they might influence susceptibility to, or evolution of, cervical lesion. Patients and methods: Forty cervical biopsies patients with low-grade cervical intraepithelial lesion and thirty-two with normal cervix were stained by immunohistochemistry for topoisomerase IIá and active caspase-3 and were investigated for the presence of HPV on exfoliated cells by general primer GP5+/6+ PCR amplification of DNA. These findings were correlated with clinicopathological features of the patients including their clinical outcome after twelve months. Subjects provided written informed consent. Results: Low-grade CIN patients as a group had a significantly higher expression of topoisomerase II alpha compared to controls, without correlation to disease outcome at 12 months. Caspase-3 was expressed in 42.5% of CIN patients and in 15.63% without disease, and most of women without caspase-3 receded cervical lesion. HPV DNA testing was positive in 65% of the patients with cervical lesion, and in 59.4% of the control group and was not associated to the expression of topoisomerase IIá or active caspase-3. In the presence of a positive HPV DNA testing, women with cervical lesion had a significantly higher expression of topoisomerase II alpha compared to controls. Conclusion: Topoisomerase II alpha and active caspase-3 might be useful diagnostic and prognostic markers in low-grade cervical lesions, delaying a better follow-up. / CNPq: 134106/2005-9 / TEDE / BV UNIFESP: Teses e dissertações

Les ADN topoisomérases de type 2 (Top2) sont des protéines universelles et essentielles à la vie cellulaire. Elles ont pour fonction de réguler de manière fine l’équilibre topologique de l’ADN. Chez l’homme, les Top2 (HsTop2) sont des cibles thérapeutiques de première importance en oncologie. Des molécules telles que l’étoposide et la doxorubicine font partie des traitements anticancéreux les plus utilisés en clinique à l’heure actuelle, elles agissent en stabilisant les complexes Top2/ADN/inhibiteurs qui sont transformés en lésions permanentes dont l’accumulation entraîne la mort cellulaire. Mon travail de thèse porte sur la caractérisation fonctionnelle et structurale des isoformes des HsTop2 entières en complexe avec des composés thérapeutiques. Mon projet de thèse comporte trois axes principaux et complémentaires : (i) La production et la caractérisation fonctionnelle in vitro des isoformes recombinantes. Nous avons optimisé et simplifié les protocoles de surexpression des deux isoformes dans la levure et avons également mis au point leur expression dans les cellules de mammifère. (ii) une étude structurale par cryo-microscopie électronique sur les HsTop2 entières en complexe avec des composés thérapeutiques et des oligonucléotides. Ces premiers travaux ont permis l’obtention d’une carte tridimensionnelle de l’enzyme entière qui servira de base pour l’étude de l’architecture des HsTop2 au sein de complexes protéiques de plus grande taille. (iii) des expériences de protéomique chimique permettant de mettre à jour les cibles secondaires potentielles de l’étoposide et de la doxorubicine dans des extraits cellulaires de lignées cancéreuses. / The type 2 DNA topoisomerases are universal proteins essential for cell survival. These enzyme fine tune the topological equilibrium of the DNA in cells. The human proteins are major targets of therapeutics drugs used in oncology. Molecules like etoposide and doxorubicin are among the most effective anticancer drugs used in chemotherapy treatments; they form stable Top2/DNA/drugs complexes which are then transformed in permanent DNA damages. This thesis project focuses on the functional and structural characterization of these complexes using 3 main strategies:(i) the production and the in vitro functional characterization of the recombinant isoforms. We optimized and simplified the production and purification procedures and overexpressed both isoforms in mammalian cells. (ii) a structural study by cryoelectron microscopy on the full enzyme complex with DNA and therapeutic drugs. We obtained a 3D density map that will be used for further studies on the architecture of large HsTop2 associated complexes. (iii) ChemoProteomic experiments on cancer cell lines to highlight the potential etoposide and doxorubicin secondary targets.