Please use this identifier to cite or link to this item: http://hdl.handle.net/10316/32378
Title: Vitamina C e cancro. Estudo experimental
Authors: Lourenço, Ana Salomé dos Santos Pires 
Orientador: Botelho, Maria Filomena
Ribeiro, Ana Bela Sarmento
Keywords: vitamina C; cancro colorretal; efeito citotóxico; efeito quimiossensibilizante; vitamin C; colorectal cancer; cytotoxix effect; chemo-sensitizing effect
Issue Date: 13-Feb-2017
Citation: LOURENÇO, Ana Salomé dos Santos Pires - Vitamina C e cancro : estudo experimental. Coimbra : [s.n.], 2017. Tese de doutoramento. Disponível na WWW: http://hdl.handle.net/10316/32378
Project: info:eu-repo/grantAgreement/FCT/SFRH/SFRH/BD/75300/2010/PT 
info:eu-repo/grantAgreement/FCT/COMPETE/132965/PT 
info:eu-repo/grantAgreement/FCT/5876/147358/PT 
Abstract: O cancro colorretal é o terceiro tipo de cancro mais incidente e representa 9,7% da incidência de cancro em todo o mundo. A taxa de mortalidade tem diminuído, principalmente nos países ocidentais, devido aos programas de rastreio e consequente deteção da doença em estádios precoces, assim como, à disponibilidade de terapias mais eficazes. A quimioterapia mantém-se uma das principais opções terapêuticas contra esta neoplasia. No entanto, esta modalidade de tratamento convencional mostra-se muitas vezes insuficientemente eficaz e altamente associada a efeitos adversos limitantes da prossecução dos tratamentos. O potencial da vitamina C na terapia do cancro há muito que tem sido avaliado, pelas suas propriedades pró-oxidantes e pelo seu potencial em reduzir os efeitos adversos relacionados com o tratamento do cancro. Dada a polarização de corrente de pensamento entre o ceticismo relativo à eficácia terapêutica de concentrações farmacológicas de vitamina C e as evidências altamente sugestivas dos seus efeitos benéficos no tratamento do cancro, há uma necessidade eminente de conhecimento dos mecanismos específicos pelos quais a vitamina C atua contra a célula tumoral. Atuais revisões sistemáticas têm sugerido uma reduzida robustez dos estudos pré-clínicos e dos ensaios clínicos realizados com concentrações farmacológicas de vitamina C. Esta é a razão que motivou a realização deste trabalho experimental, cujo objetivo é avaliar o potencial da vitamina C, na forma de ácido ascórbico (AA), no tratamento do cancro colorretal. A citotoxicidade da vitamina C em três linhas celulares humanas de cancro colorretal, com diferentes perfis genéticos, foi avaliada e o seu mecanismo anti-tumoral foi estudado. Para tal, inúmeras técnicas de biologia celular e molecular foram utilizadas, entre elas, o ensaio da sulforrodamina B, a citometria de fluxo, a microscopia ótica, o western blot, a marcação radioativa e os estudos de influxo. Adicionalmente, pretendeu-se averiguar o efeito quimiossensibilizante da vitamina C in vitro, com a implementação de um modelo de combinação de fármacos baseado num desenho experimental em raio. O modelo animal heterotópico de cancro colorretal foi desenvolvido para avaliação do potencial terapêutico e sinérgico da vitamina C in vivo. Os estudos de citotoxicidade mostraram que o ácido ascórbico induziu efeitos anti-proliferativos, citotóxicos e genotóxicos nas três linhas celulares de cancro colorretal. As vias de morte ativadas pelo ácido ascórbico foram dependentes da sensibilidade das linhas celulares ao stresse oxidativo e da intensidade do estímulo oxidativo. O ácido ascórbico foi igualmente capaz de induzir morte celular por um mecanismo independente de espécies reativas de oxigénio, de caspases e da proteína P53. Elevadas concentrações de ácido ascórbico permitiram ainda reverter a quimiorresistência da linha celular LS1034. O modelo de combinação de fármacos mostrou que concentrações elevadas de ácido ascórbico sensibilizam a célula de cancro colorretal ao efeito do 5-FU, da oxaliplatina e do irinotecano. Os efeitos anti-proliferativos mais notórios ocorreram quando o ácido ascórbico estava presente em maior proporção. A combinação de ácido ascórbico com oxaliplatina revelou ser a mais promissora, facto corroborado pelos resultados dos estudos in vivo. Os estudos in vivo mostraram ainda que concentrações farmacológicas de ácido ascórbico inibiram o crescimento de xenotransplantes de cancro colorretal e potenciaram os efeitos citotóxicos da oxaliplatina e do irinotecano. Este trabalho de investigação fundamental reforça o potencial da vitamina C no tratamento do cancro colorretal. De facto, concentrações farmacológicas deste nutriente medeiam diferentes mecanismos de ativação de morte da célula tumoral e sensibilizam-na para o efeito da quimioterapia. Este conhecimento mais aprofundado dos mecanismos de ação da vitamina C e a expectável rigorosa avaliação da sua eficácia clínica poderão contribuir para o estabelecimento de protocolos de tratamento mais eficazes, economicamente sustentáveis, de baixa toxicidade e que contribuam para a melhoria da qualidade de vida do doente oncológico. Colorectal cancer is the third most frequent type of cancer worldwide, representing an incidence of 9.7%. The mortality rate has decreased, especially in Western countries due to screening programs implementation and the subsequent detection of the disease in early stages, as well as the availability of more effective therapies. However, chemotherapy remains a major therapeutic option against this type of cancer. This conventional treatment modality often displays insufficient effectiveness and highly adverse effects limiting the pursuit of treatments. The potential of vitamin C in cancer therapy has long been evaluated by its pro-oxidant properties and its potential to reduce the adverse effects related to the cancer treatment. Given the division of opinions between the skepticism around the therapeutic efficacy of pharmacological concentrations of vitamin C and the highly suggestive evidence of their beneficial effects in the treatment of cancer, there is an imminent need for knowledge of the specific mechanisms by which vitamin C acts against tumor cell. Current systematic reviews have suggested a reduced robustness of preclinical studies and clinical trials with pharmacologic concentrations of vitamin C. This is the reason that motivated this experimental study, which aims to assess the potential of vitamin C, in the form of ascorbic acid (AA), for the treatment of colorectal cancer. The cytotoxicity of vitamin C in three human colorectal cancer cell lines, with different genetic profiles, was evaluated and its antitumor mechanism was studied. For this purpose, several cellular and molecular biology techniques have been used, such as sulforhodamine B assay, flow cytometry, light microscopy, western blot analysis, radioactive labeling and influx studies. In addition, it sought to determine the chemosensitive effect of vitamin C in vitro, with the implementation of a model of drugs combination based on a ray experimental design. The heterotopic animal model of colorectal cancer has been developed for evaluation of therapeutic and synergistic potential of vitamin C in vivo. Cytotoxicity studies showed that ascorbic acid induces anti-proliferative, cytotoxic and genotoxic effects in cells of the three cell lines of colorectal cancer. The cell death pathways activated by ascorbic acid are dependent on the sensitivity of cell lines to oxidative stress and oxidative stimulus intensity. Ascorbic acid is also capable of inducing cell death through a mechanism independent of reactive oxygen species, caspases and P53. High concentration of ascorbic acid also allowed to reverse the chemoresistance of LS1034 cell line. The model of drugs combination showed that high concentrations of ascorbic acid sensitize the colorectal cancer cell to the effects of 5-FU, oxaliplatin and irinotecan. The most notorious anti-proliferative effects were observed when ascorbic acid is present in greater proportion. The combination of ascorbic acid and oxaliplatin was shown to be the most promising combination, fact corroborated by the results of in vivo studies. In vivo studies have shown that pharmacological concentrations of ascorbate inhibit the growth of colorectal cancer xenografts and potentiate the cytotoxic effects of oxaliplatin and irinotecan. This fundamental research work reinforces the potential of vitamin C in the treatment of colorectal cancer. In fact, pharmacological concentrations of this nutrient mediate different mechanisms of tumor cell death activation and sensitize it to the effect of chemotherapy. This deeper understanding of vitamin C mechanisms of action and the expected rigorous evaluation of its clinical effectiveness may contribute to the establishment of more effective treatment protocols, economically sustainable, with low toxicity and that contribute to the improvement of cancer patient's quality of life.
Colorectal cancer is the third most frequent type of cancer worldwide, representing an incidence of 9.7%. The mortality rate has decreased, especially in Western countries due to screening programs implementation and the subsequent detection of the disease in early stages, as well as the availability of more effective therapies. However, chemotherapy remains a major therapeutic option against this type of cancer. This conventional treatment modality often displays insufficient effectiveness and highly adverse effects limiting the pursuit of treatments. The potential of vitamin C in cancer therapy has long been evaluated by its pro-oxidant properties and its potential to reduce the adverse effects related to the cancer treatment. Given the division of opinions between the skepticism around the therapeutic efficacy of pharmacological concentrations of vitamin C and the highly suggestive evidence of their beneficial effects in the treatment of cancer, there is an imminent need for knowledge of the specific mechanisms by which vitamin C acts against tumor cell. Current systematic reviews have suggested a reduced robustness of preclinical studies and clinical trials with pharmacologic concentrations of vitamin C. This is the reason that motivated this experimental study, which aims to assess the potential of vitamin C, in the form of ascorbic acid (AA), for the treatment of colorectal cancer. The cytotoxicity of vitamin C in three human colorectal cancer cell lines, with different genetic profiles, was evaluated and its antitumor mechanism was studied. For this purpose, several cellular and molecular biology techniques have been used, such as sulforhodamine B assay, flow cytometry, light microscopy, western blot analysis, radioactive labeling and influx studies. In addition, it sought to determine the chemosensitive effect of vitamin C in vitro, with the implementation of a model of drugs combination based on a ray experimental design. The heterotopic animal model of colorectal cancer has been developed for evaluation of therapeutic and synergistic potential of vitamin C in vivo. Cytotoxicity studies showed that ascorbic acid induces anti-proliferative, cytotoxic and genotoxic effects in cells of the three cell lines of colorectal cancer. The cell death pathways activated by ascorbic acid are dependent on the sensitivity of cell lines to oxidative stress and oxidative stimulus intensity. Ascorbic acid is also capable of inducing cell death through a mechanism independent of reactive oxygen species, caspases and P53. High concentration of ascorbic acid also allowed to reverse the chemoresistance of LS1034 cell line. The model of drugs combination showed that high concentrations of ascorbic acid sensitize the colorectal cancer cell to the effects of 5-FU, oxaliplatin and irinotecan. The most notorious anti-proliferative effects were observed when ascorbic acid is present in greater proportion. The combination of ascorbic acid and oxaliplatin was shown to be the most promising combination, fact corroborated by the results of in vivo studies. In vivo studies have shown that pharmacological concentrations of ascorbate inhibit the growth of colorectal cancer xenografts and potentiate the cytotoxic effects of oxaliplatin and irinotecan. This fundamental research work reinforces the potential of vitamin C in the treatment of colorectal cancer. In fact, pharmacological concentrations of this nutrient mediate different mechanisms of tumor cell death activation and sensitize it to the effect of chemotherapy. This deeper understanding of vitamin C mechanisms of action and the expected rigorous evaluation of its clinical effectiveness may contribute to the establishment of more effective treatment protocols, economically sustainable, with low toxicity and that contribute to the improvement of cancer patient's quality of life.
Description: Tese de doutoramento em Engenharia Biomédica, apresentada à Faculdade de Ciências e Tecnologia da Universidade de Coimbra
URI: http://hdl.handle.net/10316/32378
Rights: openAccess
Appears in Collections:FCTUC Ciências da Vida - Teses de Doutoramento

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