Submitted by Edineia Teixeira (edineia.teixeira@unioeste.br) on 2018-02-23T18:26:15Z No. of bitstreams: 2 Jose_santos2017.pdf: 2362954 bytes, checksum: 3cd8da60eb3efb9e2ed240ef51fa9a23 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-02-23T18:26:15Z (GMT). No. of bitstreams: 2 Jose_santos2017.pdf: 2362954 bytes, checksum: 3cd8da60eb3efb9e2ed240ef51fa9a23 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-08-01 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Lung cancer is highly lethal and smoking is an important risk factor. Non-small cell tumor is the most common, less aggressive type of lung carcinoma and treatment involves surgery, chemotherapy, and radiation therapy. However, late diagnosis, due to the absence of signs and symptoms in the early stages of the disease, makes the survival rate low. In this sense, the search for new chemical substances with characteristics of selectivity, effectiveness and low toxicity have been investigated in the treatment of cancer. Studies show that sericin, a protein extracted from silkworm cocoons, exhibits anti-tumor and anticarcinogenic activity in colon and skin cancer cells, stimulating apoptosis and disrupting the cell cycle, and protecting normal cells against oxidative stress and lipid peroxidation . Thus, the therapeutic potential of sericin has raised interest in evaluating its effect on non-small cell lung cancer cell line. The studies were conducted in culture, where the effects of silk protein were evaluated: on cell viability, by neutral red assays and tetrazolium - MTT cytotoxicity; in the apoptotic potential, with annexin-5 and Alexa Fluor® and Propidium Iodide assays; and in cell migration by the Wound Healing model assay. Low doses of sericin were able to increase lysosomal viability, reduce mitochondrial viability, increase apoptosis and cell migration, while high doses of sericin exponentially reduced cell migration and did not alter the rate of apoptosis / necrosis of cancer cells. Sericin is a biomaterial that causes biological effects on the cell line tested, and can be used to increase lysosomal viability, reduce mitochondrial function, increase apoptosis at low doses and inhibit high-dose cell migration. / O câncer de pulmão apresenta alta letalidade e o fumo se constitui como um importante fator de risco. O tumor de células não pequenas é o tipo de carcinoma pulmonar mais comum, menos agressivo e o tratamento envolve cirurgia, quimioterapia e radioterapia. Entretanto, o diagnóstico tardio, em função da ausência de sinais e sintomas nos estágios iniciais da doença, faz com que a taxa de sobrevivência seja baixa. Nesse sentido, a busca de novas substâncias químicas com características de seletividade, de efetividade e de baixa toxicidade tem se refletido em pesquisas que investigam o uso dessas substâncias no tratamento do câncer. Estudos mostram que a sericina, proteína extraída dos casulos do bicho-da-seda, apresenta atividade antitumoral e anticarcinogênica em células cancerosas de cólon e de pele, estimulando a apoptose e interrompendo o ciclo celular, além de proteger células normais contra estresse oxidativo e peroxidação lipídica. Assim, o potencial terapêutico da sericina suscitou o interesse em avaliar seu efeito em linhagem celular de câncer de pulmão do tipo células não pequenas. Os estudos foram conduzidos em cultura, em que foram avaliados os efeitos da proteína da seda: na viabilidade celular, por meio de ensaios pelo vermelho neutro e de citotoxicidade com o tetrazólio - MTT; no potencial apoptótico, com ensaios com anexina-5 e Alexa Fluor® e Iodeto de Propídio; e na migração celular, ensaio com o modelo Wound Healing. Baixas doses de sericina foram capazes de aumentar a viabilidade lisossomal, reduzir a viabilidade mitocondrial, aumentar a apoptose e a migração celular, enquanto altas doses de sericina reduziram exponencialmente a migração celular e não alteraram a taxa de apoptose/necrose das células cancerosas. A sericina é um biomaterial que provoca efeitos biológicos na linhagem celular testada, e pode ser utilizada para aumentar a viabilidade lisossomal, reduzir a função mitocondrial, aumentar a apoptose em baixas doses e inibir a migração celular em doses altas.

BACKGROUND: DNA damage occurs as a consequence of both direct and indirect effects of ionizing radiation. The severity of DNA damage depends on the physical characteristics of the radiation quality, e.g., the linear energy transfer (LET). There are still contrary findings regarding direct or indirect interactions of high-LET emitters with DNA. Our aim is to determine DNA damage and the effect on cellular survival induced by (223)Ra compared to (188)Re and (99m)Tc modulated by the radical scavenger dimethyl sulfoxide (DMSO). METHODS: Radioactive solutions of (223)Ra, (188)Re, or (99m)Tc were added to either plasmid DNA or to PC Cl3 cells in the absence or presence of DMSO. Following irradiation, single strand breaks (SSB) and double strand breaks (DSB) in plasmid DNA were analyzed by gel electrophoresis. To determine the radiosensitivity of the rat thyroid cell line (PC Cl3), survival curves were performed using the colony formation assay. RESULTS: Exposure to 120 Gy of (223)Ra, (188)Re, or (99m)Tc leads to maximal yields of SSB (80 %) in plasmid DNA. Irradiation with 540 Gy (223)Ra and 500 Gy (188)Re or (99m)Tc induced 40, 28, and 64 % linear plasmid conformations, respectively. DMSO prevented the SSB and DSB in a similar way for all radionuclides. However, with the α-emitter (223)Ra, a low level of DSB could not be prevented by DMSO. Irradiation of PC Cl3 cells with (223)Ra, (188)Re, and (99m)Tc pre-incubated with DMSO revealed enhanced survival fractions (SF) in comparison to treatment without DMSO. Protection factors (PF) were calculated using the fitted survival curves. These factors are 1.23 ± 0.04, 1.20 ± 0.19, and 1.34 ± 0.05 for (223)Ra, (188)Re, and (99m)Tc, respectively. CONCLUSIONS: For (223)Ra, as well as for (188)Re and (99m)Tc, dose-dependent radiation effects were found applicable for plasmid DNA and PC Cl3 cells. The radioprotection by DMSO was in the same range for high- and low-LET emitter. Overall, the results indicate the contribution of mainly indirect radiation effects for each of the radionuclides regarding DNA damage and cell survival. In summary, our findings may contribute to fundamental knowledge about the α-particle induced DNA damage.

BACKGROUND: DNA damage occurs as a consequence of both direct and indirect effects of ionizing radiation. The severity of DNA damage depends on the physical characteristics of the radiation quality, e.g., the linear energy transfer (LET). There are still contrary findings regarding direct or indirect interactions of high-LET emitters with DNA. Our aim is to determine DNA damage and the effect on cellular survival induced by (223)Ra compared to (188)Re and (99m)Tc modulated by the radical scavenger dimethyl sulfoxide (DMSO). METHODS: Radioactive solutions of (223)Ra, (188)Re, or (99m)Tc were added to either plasmid DNA or to PC Cl3 cells in the absence or presence of DMSO. Following irradiation, single strand breaks (SSB) and double strand breaks (DSB) in plasmid DNA were analyzed by gel electrophoresis. To determine the radiosensitivity of the rat thyroid cell line (PC Cl3), survival curves were performed using the colony formation assay. RESULTS: Exposure to 120 Gy of (223)Ra, (188)Re, or (99m)Tc leads to maximal yields of SSB (80 %) in plasmid DNA. Irradiation with 540 Gy (223)Ra and 500 Gy (188)Re or (99m)Tc induced 40, 28, and 64 % linear plasmid conformations, respectively. DMSO prevented the SSB and DSB in a similar way for all radionuclides. However, with the α-emitter (223)Ra, a low level of DSB could not be prevented by DMSO. Irradiation of PC Cl3 cells with (223)Ra, (188)Re, and (99m)Tc pre-incubated with DMSO revealed enhanced survival fractions (SF) in comparison to treatment without DMSO. Protection factors (PF) were calculated using the fitted survival curves. These factors are 1.23 ± 0.04, 1.20 ± 0.19, and 1.34 ± 0.05 for (223)Ra, (188)Re, and (99m)Tc, respectively. CONCLUSIONS: For (223)Ra, as well as for (188)Re and (99m)Tc, dose-dependent radiation effects were found applicable for plasmid DNA and PC Cl3 cells. The radioprotection by DMSO was in the same range for high- and low-LET emitter. Overall, the results indicate the contribution of mainly indirect radiation effects for each of the radionuclides regarding DNA damage and cell survival. In summary, our findings may contribute to fundamental knowledge about the α-particle induced DNA damage.