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Biological characterization and vectorization of new compounds with antiproliferative effects
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A quimioterapia mantem-se, ainda nos dias de hoje, o tratamento maioritário aplicado contra o cancro. No entanto, os efeitos secundários graves e a resistência por parte das células cancerígenas à terapêutica permanecem obstáculos consideráveis, pelo que a descoberta de novos fármacos revela-se uma prioridade. Neste contexto, o trabalho desenvolvido nesta dissertação teve como objectivo a caracterização completa do efeito antitumoural de um composto de cobre e a respectiva nanovectorização. Os ensaios in vitro em linhas tumorais HCT116 revelaram que o complexo apresenta valores de IC50 de 5,4 μM. A citoselectividade para células tumorais foi avaliada na linha celular de Fibroblastos, que apresentou um aumento do valor de IC50 em cerca de cinco vezes. A marcação com Hoechst 33258 de células HCT116 incubadas com 5,4 e 8,1 μM de composto não revelou fragmentação e condensação nuclear, características do mecanismo de apoptose. Estes resultados foram confirmados pela análise por citometria de fluxo com marcação dupla com anexina V-FITC e iodeto de propídio, em que cerca de 80% das células HCT116 estão viáveis. Realizaram-se, ainda, estudos de interacção com o DNA, para determinar a afinidade do complexo para esta molécula. A constante de afinidade, determinada por titulações UV, apresentou um valor de 4.17x103 M-1, diversas ordens de magnitude inferior ao valor para a Doxorrubicina. Dado que não foi observado retardamento da mobilidade electroforética da forma superenrolada do DNA plasmídico, com o aumento da concentração de composto, a interacção deste com o DNA ocorre, possivelmente, pela ligação ao sulco menor do DNA. O ensaio de progressão do ciclo celular mostrou que o composto não influencia este processo. Ambos os ensaios de interacção com a proteína BSA, revelaram que o composto tem grande afinidade para proteínas, cuja contante de afinidade e número de ligações apresentaram o valor de 4,94x103 M-1 e 0,63, respectivamente. A vectorização do composto em nanopartículas de ouro revelou uma diminuição mais acentuada da viabilidade celular (em cerca de 20%), possivelmente devido a diferentes mecanismos de intercalação. Pelo ensaio de proteómica comparativa foi possível observar, entre outros, que a exposição ao composto resulta numa sub-expressão de proteínas associadas ao citoesqueleto, uma sub-expressão de proteínas antioxidantes e uma sobre-expressão da proteína de resposta ao stress.
Nowadays, chemotherapy is still the majority of the cancer treatments. However, the severe side effects and the drug resistance by cancer cells remain giant obstacles, so much that the discovery of new drugs is still a major priority. In this context, the work developed on this thesis aimed the full characterization of the antitumor effect of a copper compound and the respective vectorization. In vitro assay on HCT116 cell line showed that the compound had an IC50 of 5.4 μM. Compound’s cytoselectivity to tumoral cells was evaluated on primary cell line Fibroblasts, which presented an IC50 value 5-fold higher than the one observed for HCT116. Hoechst 33258 staining in HCT116, incubated with 5.4 and 8.1 μM of compound, didn’t reveal nuclear fragmentation or condensation, hallmarks of the apoptosis mechanism. These results were confirmed by flow cytometry with an Annexin V-FITC and PI double staining of HCT116 cells, in which around 80% of cells were viable. Were also performed DNA interaction studies in order to calculate an affinity constant, which was 4.17x103 M-1, several orders of magnitude lower than that for Doxorubicin. The electrophoretic mobility shift assay showed no shifting of the supercoiled pDNA form with increased compound concentration, and together, these results point to an interaction with DNA through weak minor groove binding. Cell cycle progression assay revealed that the compound has no interference in this process. Both BSA interaction assays showed a strong affinity of the compound to proteins, which binding constant and the number of binding sites presented a value of 4.94x103 M-1 e 0.63, respectively. Nanoparticle vectorization presented a greater reduction in cell viability (at around 20%), possibly due to different internalization mechanisms. Proteomics assay revealed that the exposure to compound results in under expression of cytoskeleton associated proteins, under expression of antioxidant proteins and over expression of stress related proteins.
Nowadays, chemotherapy is still the majority of the cancer treatments. However, the severe side effects and the drug resistance by cancer cells remain giant obstacles, so much that the discovery of new drugs is still a major priority. In this context, the work developed on this thesis aimed the full characterization of the antitumor effect of a copper compound and the respective vectorization. In vitro assay on HCT116 cell line showed that the compound had an IC50 of 5.4 μM. Compound’s cytoselectivity to tumoral cells was evaluated on primary cell line Fibroblasts, which presented an IC50 value 5-fold higher than the one observed for HCT116. Hoechst 33258 staining in HCT116, incubated with 5.4 and 8.1 μM of compound, didn’t reveal nuclear fragmentation or condensation, hallmarks of the apoptosis mechanism. These results were confirmed by flow cytometry with an Annexin V-FITC and PI double staining of HCT116 cells, in which around 80% of cells were viable. Were also performed DNA interaction studies in order to calculate an affinity constant, which was 4.17x103 M-1, several orders of magnitude lower than that for Doxorubicin. The electrophoretic mobility shift assay showed no shifting of the supercoiled pDNA form with increased compound concentration, and together, these results point to an interaction with DNA through weak minor groove binding. Cell cycle progression assay revealed that the compound has no interference in this process. Both BSA interaction assays showed a strong affinity of the compound to proteins, which binding constant and the number of binding sites presented a value of 4.94x103 M-1 e 0.63, respectively. Nanoparticle vectorization presented a greater reduction in cell viability (at around 20%), possibly due to different internalization mechanisms. Proteomics assay revealed that the exposure to compound results in under expression of cytoskeleton associated proteins, under expression of antioxidant proteins and over expression of stress related proteins.
Descrição
Tese de mestrado em Biologia Molecular e Genética, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2015
Palavras-chave
Cancro Complexo de cobre Apoptose Ciclo celular Nanovectorização Teses de mestrado - 2015