Submitted by Haroudo Xavier Filho (haroudo.xavierfo@ufpe.br) on 2016-04-05T17:59:08Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE_SINARA MONICA VITALINO DE ALMEIDA_FINAL UNIFICADO BIBLIOTECA.pdf: 9803056 bytes, checksum: 68bd08234fdac2b45bf400b3ebd62957 (MD5) / Made available in DSpace on 2016-04-05T17:59:08Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE_SINARA MONICA VITALINO DE ALMEIDA_FINAL UNIFICADO BIBLIOTECA.pdf: 9803056 bytes, checksum: 68bd08234fdac2b45bf400b3ebd62957 (MD5) Previous issue date: 2015-07-23 / CAPES, CNPq / O câncer é, sem sombra de dúvidas, a doença mais temida pela população, em razão da sua alta incidência e elevadas taxas de mortalidade para determinados tipos da doença. Nas últimas décadas, os pesquisadores têm obtido avanços significativos no entendimento da patogênese, nas características e nas terapias do câncer. A quimioterapia é frequentemente o tratamento escolhido para muitos tipos de câncer e por este motivo a pesquisa por novos agentes quimioterápicos constitui um dos alicerces na luta contra o câncer. Os intercaladores orgânicos são compostos poliaromáticos que podem se inserir entre pares de bases adjacentes da dupla fita de DNA e inibir a síntese de ácido nucléico in vivo, essa propriedade é comumente observada em drogas anticâncer usadas na terapia clínica. Por isto, a descoberta de novos intercaladores do DNA tem sido considerada uma abordagem prática e um número expressivo de moléculas tem sido avaliado quanto às suas propriedades intercaladoras. Neste trabalho foram sintetizados novos agentes anticâncer tendo como molécula de partida o anel acridina. Foram sintetizados e caracterizados oito novos derivados da série 2-acridin-9-il-metileno-N-fenil-hidrazina-carbotioamida (3a-3h) com diferentes substituintes na porção fenil (não substituídos ou com substituintes elétrondoadores ou elétron-retiradores) e dois novos derivados da série 3-(acridin-9-il)-n-benzilideno-2- cianoacrilohidrazidas (AMTAC-01 e AMTAC-02). Os compostos foram avaliados quanto às suas propriedades intercaladoras ao ctDNA in vitro e atividades antiproliferativas contra linhagens de células tumorais de mama (MCF-7), ovário resistente a múltiplas drogas (NCI-ADR/RES), pulmão (NCI-H460), próstata (PC-3), cólon (HT29), ovário (OVCAR-03), rim (786-0), leucemia (K562) e glioma (U251). Foram investigadas alterações morfológicas induzidas por tratamento de células MCF-7 com o composto mais ativo da série 2-acridin-9-il-metileno-N-fenil-hidrazinacarbotioamida (3a) através de microscopias eletrônicas de transmissão, varredura e da análise de exposição de fosfatidilserina e fragmentação de DNA. Além disso, a atividade de inibição da topoisomerase IIa dos derivados 3-(acridin-9-il)-n-benzilideno-2-cianoacrilohidrazidas foi verificada e a ligação com ctDNA foi estudada por meio de espectroscopia de absorção em fluorescência. Todos os derivados produzidos das duas séries apresentaram interação com o DNA. Após o contato com DNA foram verificados efeitos hipercrômicos e hipocrômicos, bem como mudanças para o vermelho ou azul nos espectros de absorbância. Essas modificações são preditivas de formação de complexo entre DNA e derivado. As constantes de ligação calculadas estão entre 1.74 x 104 e 1.0 x 106 M-1 para os derivados 3a-3h e entre 2.3-2.5 x 106 M-1 para os AMTAC’s. Estes valores indicam alta afinidade pelos pares de base do DNA. Da série 2-acridin- 9-il-metileno-N-fenil-hidrazina-carbotioamida o composto mais eficiente para ligação in vitro com o DNA foi o derivado cloro-substituído (3f), enquanto o composto mais ativo no teste antiproliferativo foi o derivado não substituído na porção tiossemicarbazona (3a). Os valores de concentração letal para 50 % do número inicial de células para o derivado 3a contra as linhagens NCI-H460, MCF-7, U251, NCI-ADR/RES, HT-29 e PC-3 foram 43.41, 60.26, 68.93, 70.2, 70.24 e 72.95 μM, respectivamente. As análises por microscopias eletrônicas de varredura e transmissão de células MCF-7 tratadas com 60 μM do derivado 3a demonstraram alterações ultramorfológicas indicativas de autofagia: vacúolos com dupla membrana. Além disso, os derivados AMTAC-01 e AMTAC-02 foram mais ativos contra as linhagens tumorais de próstata e melanoma, respectivamente. Ambos os derivados apresentaram atividade inibidora topoisomerase IIa na concentração de 50 μM. Os resultados indicam que uma ligação eficiente ao DNA é uma condição necessária para atividade antitumoral e que os novos derivados híbridos de acridina apresentaram promissoras atividades antiproliferativa, ligadora do DNA e inibição da topoisomerase. / People fear cancer more than any other serious illness which can be explained by the high incidence and mortality rates for some types of cancer. In the last decades, significant advances were obtained regarding cancer pathogenesis, features and therapies. Chemotherapy is often the treatment of choice for many types of cancer and the search for new chemotherapeutic agents still plays a major role in the fight against cancer. Organic intercalators are poliaromatic compounds that are able to insert into DNA double strands and inhibit in vivo acid nucleic synthesis. This characteristic is, in general, observed in anticancer drugs, hence the discovery and development of new DNA intercalators has been considered a practical approach and a number of intercalators have been recently reported. In this work, new anticancer agents were synthetized based on acridine nucleus for structural modification using substituted thiosemicarbazide moieties. It were synthetized eight new (Z)-2-(acridin-9- ylmethylene)-N-phenylhydrazinecarbothioamide derivatives (3a-3h) presenting different substituents on phenyl ring (non-substituted and electron-donating or -withdrawing) and two new 3-(acridin-9-yl)-N-benzylidene-2-cyanoacrilohydrazide derivatives (AMTAC-01 and AMTAC-02). In vitro ctDNA interaction was assayed and antiproliferative activity was evaluated against cancer cell lines of glioma (U251), breast (MCF-7), ovary expressing phenotype multiple drugs resistance (NCI-ADR/RES), kidney (786–0), lung (NCI-H460), prostate, (PC-3), ovary (OVCAR-03), colon adenocarcinoma (HT-29) and chronic myeloid leukemia (K-562). It was investigated ultramorphological changes induced by 3a treatment on MCF-7 cells by transmission and scanning electron microscopies, besides the evaluation of phosphatidylserine externalization and DNA fragmentation. Topoisomerase IIa inhibitory activity of AMTAC’s was evaluated. ctDNA binding properties were performed with calf thymus DNA (ctDNA) by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated by addition of ctDNA to the derivatives. These spectroscopic alterations indicated formation of derivative-DNA complex. The calculated binding constants ranged from 1.74 x 104 to 1.0 x 106 M-1 for 3a-3h derivatives and 2.3-2.5 x 106 M-1 for AMTAC’s compounds. These values mean that the new acridine derivatives have high affinity to ctDNA. From (Z)-2-(acridin-9- ylmethylene)-N-phenylhydrazinecarbothioamide serie, the most efficient compound in in vitro binding to ctDNA was 3f, while the most active compound in antiproliferative assay was 3a. Regarding lethal concentration (LC50), compound 3a was lethal to NCI-H460, MCF-7, U251, NCI-ADR/RES, HT-29 and PC-3 cells on the respective concentrations: 43.41, 60.26, 68.93, 70.2, 70.24 and 72.95 μM. Scanning and transmission electron microscopies revealed that treatment with 60 μM of 3a induces morphological changes in MCF-7 cells indicating autophagy, such as vacuole with double membrane. On the other hand, antiproliferative assay demonstrated that AMTAC-01 and AMTAC-02 were most active against prostate and melanoma tumor cell lines, respectively. Both derivatives displayed potent topoisomerase IIα inhibitory activity at 50 μM. Taking together, these results indicates that an efficient binding is a necessary condition for antiproliferative activity. The new acridine hybrid derivatives showed promising DNA binding, antiproliferative against cancer cells and inhibitory topoisomerase activity.

Human response to ionising radiation (IR) shows a wide variation. This is most clearly seen in the radiation-response of cells as measured by frequencies of chromosomal aberrations. Different frequencies of IR-induced aberrations can be conveniently observed in phytohaemagglutin-stimulated peripheral blood T-lymphocytes from both normal individuals and sporadic cancer cases, in either metaphase chromosomes or as micronuclei in the following cell cycle. Metaphase cells show frequent chromatid breaks, defined as chromatid discontinuities or terminal deletions, if irradiated in the G 2 -phase of the cell cycle. It has been shown that the frequency of chromatid breaks in cells from approximately 40% of sporadic breast cancer patients, are significantly higher than in groups of normal individuals. This suggests that elevated radiation-induced chromatid break frequency may be linked with susceptibility to breast cancer. It is known that chromatid breaks are initiated by a double strand break (DSB), but it appears that the two are linked only indirectly as repair kinetics for DSBs and chromatid breaks do not match. Therefore, the underlying causes of the wide variation in frequencies of chromatid breaks in irradiated T-lymphocytes from different normal individuals and from sporadic breast cancer cases are still unclear but it is unlikely to be linked directly to DSB rejoining. My research has focused on the mechanism through which chromatid breaks are formed from initial DSBs. The lack of a direct association suggested that a signalling process might be involved, connecting the initial DSB and resulting chromatid break. The signal model, suggested that the initial DSB is located within a chromatin loop that leads to an intra- or interchromatid rearrangement resulting in incomplete mis-joining of chromatin ends during the decatenation of chromatids during G 2 . It was therefore proposed that topoisomerase II alpha (topo IIα) might be involved, mainly because of its ability to incise DNA and its role in sister chromatid decatenation. During my PhD research I have used a strategy of altering topo II activity or expression and studying whether this alters IR-induced chromatid break frequency. The first approach involved cell lines that varied in topo IIα expression. The frequency of IR-induced chromatid breaks was found to correlate positively with topo IIα expression level, as measured in three different cell lines by immunoblotting, i.e. two cell lines with lower topo IIα expression exhibited lower chromatid break frequency. Topo II activity in these three cell lines was also estimated indirectly by the ability of a topo IIα poison to activate the G 2 /M checkpoint, and this related well with topo IIα expression. A second approach involved ‘knocking down’ topo IIα protein expression by silencing RNA (siRNA). Lowered topo IIα expression was confirmed by immunoblotting and polymerase chain reaction. SiRNA-lowered topo IIα expression correlated with a decreased IR-induced chromatid break frequency. In a third series of experiments cells were treated with ICRF-193, a topo IIα catalytic inhibitor. It was shown that inhibition of topo IIα also significantly reduced IR-induced chromatid breaks. I also showed that lowered chromatid break frequency was not due to cells with high chromatid break frequencies being blocked in G 2 as the mitotic index was not altered significantly in cells with lowered topo IIα expression or activity. These experiments show that topo IIα is involved in IR-induced chromatid break formation. The final experiments reported here attempted to show how topo II might be recruited in the process of forming IR-induced chromatid breaks. Hydrogen peroxide was used as a source of reactive oxygen species (reported to poison topo IIα) and it was shown that topo IIα under these conditions is involved in the entanglement of metaphase chromosomes and formation of chromatin ‘dots’ as well as chromatid breaks. Experiments using atomic force microscopy attempted to confirm these dots as excised chromatin loops. The possible role of topo IIα in both radiation- and hydrogen peroxide-induced primary DNA damage was also tested. It was shown that topo IIα does not affect radiation-induced DSBs, even though it does affect chromatid break frequency. Also, topo IIα does not affect hydrogen peroxide-induced DNA damage at low doses. The results support the idea that topo IIα is involved in the conversion of DSBs to chromatid breaks after both irradiation and treatment with hydrogen peroxide at a low concentrations. I have demonstrated that topo IIα is involved in forming IR-induced chromatid breaks, most likely by converting the initial DSBs into chromosomal aberrations as suggested by the signal model.