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Produtos naturais na metabolização de fármacos quimioterápicos
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Atualmente, o cancro é uma das doenças mais frequentes no mundo, sendo a quimioterapia o tratamento mais comum. Muitos doentes, durante a quimioterapia convencional, recorrem ao tratamento suplementar com produtos naturais e suplementos alimentares considerados úteis na cura do cancro, contudo existem poucos estudos que comprovem a sua eficácia. O objetivo deste estudo é investigar produtos naturais que possam influenciar a atividade de dois fármacos comuns na quimioterapia do cancro: o tamoxifeno e a cisplatina. Sendo o tamoxifeno um pró-fármaco, torna-se também necessário estudar o efeito dos produtos naturais na sua metabolização. Para isso foram efetuados estudos de citotoxicidade dos dois fármacos na presença de produtos naturais provenientes da dieta: quercetina, rutina (forma glicosilada de quercetina) e produtos resultantes da digestão de Spirulina platensis. Estudou-se também a influência da albumina do soro bovino (BSA), uma proteína transportadora de solutos no soro, na citotoxicidade do tamoxifeno. Estes estudos demonstram que a atividade do tamoxifeno nas células HeLa aumenta na presença de quercetina e diminui na presença de rutina, enquanto nas células MCF-7 a sua atividade diminui na presença de produtos da digestão de S. platensis. Para além disso, em ambas as linhas celulares, a atividade do tamoxifeno diminui acentuadamente na presença da BSA, provavelmente devido a ligação do fármaco à proteína. No caso da cisplatina, a sua atividade diminui na presença de quercetina e de produtos da digestão de S. platensis nas células HeLa e diminui na presença de quercetina, rutina e produtos da digestão de S. platensis nas células MCF-7. Para uma melhor compreensão dos mecanismos subjacentes à interação entre os fármacos e produtos naturais, foram efetuados estudos complementares - estudos de proliferação, de metabolização in vitro e de alterações na estrutura celular por espetroscopia de FTIR. Os estudos de proliferação demonstram que nas células HeLa ambos os fármacos têm efeito no mecanismo de invasão das células tumorais. A interação do tamoxifeno com a rutina e produtos da digestão de S. platensis e a interação da cisplatina com a quercetina e rutina têm efeito no aumento da aderência e subsequente proliferação das células HeLa. O mesmo efeito foi observado na combinação da cisplatina com a rutina nas células MCF-7. Nos estudos de metabolização in vitro pelos citocromos P450 e pelas células Caco-2 e HepG2, não foram detetados metabolitos do tamoxifeno, pelo que não foi possível realizar o estudo da interação dos produtos naturais na metabolização do tamoxifeno. Para além disso, quando presente em amostras biológicas, tornou-se difícil a análise do tamoxifeno por HPLC-DAD e FTIR, provavelmente devido à sua ligação à albumina presente no soro e também devido à sua hidrofobicidade. Os estudos efetuados por espetroscopia de FTIR demonstram que o efeito dos fármacos e da combinação destes com os produtos naturais leva à morte celular por apoptose, através da fragmentação do DNA e do rearranjo na composição dos lípidos membranares, sendo estes os processos necessários para a formação de corpos apoptóticos. Os mecanismos de interação mais prováveis estão associados com a capacidade dos produtos naturais interferirem nas vias de sinalização dos fármacos, atenuando ou estimulando a indução da apoptose. Em conclusão, a quercetina, a rutina e os produtos da digestão de S. platensis alteram a ação celular do tamoxifeno e da cisplatina em várias linhas celulares tumorais. Tendo em conta que o mecanismo de interação com o tamoxifeno pode estar relacionado com a sua metabolização, é necessário que se desenvolvam novos métodos para testar esta hipótese.
Currently, cancer is one of the most frequent disease in the world, with chemotherapy being the most common type of treatment. Many patients, during conventional chemotherapy, choose to use a supplemental treatment with natural products and dietary supplements claiming health benefits for cancer treatment, however there are few studies that prove their efficacy. The main aim of this study is to investigate several natural products that may affect the activity of two common chemotherapeutic drugs like tamoxifen and cisplatin. Since tamoxifen is a prodrug, it’s also necessary to study the effect of natural products on his metabolism. For this, cytotoxicity assays of two chemotherapy drugs were performed in the presence of natural compounds derived from diet: quercetin, rutin (glycosylated form of quercetin) and the products resulting from Spirulina platensis digestion. The influence of bovine serum albumin (BSA), a serum solute carrier protein, on the cytotoxicity of tamoxifen was also studied. This assays showed that the activity of tamoxifen in HeLa cell line is influenced by the flavonoids, increasing the cytotoxic effect in the presence of quercetin and decreasing in the presence of rutin. While, in MCF-7 cell line the activity of tamoxifen decreases in the presence of the products resulting from S. platensis digestion. In addition, in both cell lines, the activity of tamoxifen declines significantly in the presence of BSA, probably due to the binding of the drug to the protein. In the case of cisplatin, the cytotoxic activity decreases in the presence of quercetin and products of S. platensis digestion in HeLa cell line and decreases in the presence of quercetin, rutin and products of S. platensis digestion in MCF-7 cell line. For a better comprehension of the mechanisms underlying the interaction between drug and natural products several studies were performed – proliferation, in vitro metabolism and cellular structure changes by FTIR spectroscopy. Proliferation studies demonstrate that in HeLa cell line both drugs have an effect on the invasion mechanism used by cancer cells. The interaction of tamoxifen with rutin and S. platensis digestion products and the interaction of cisplatin with quercetin and rutin have an effect increasing adhesion and subsequent proliferation of HeLa cell line. The same effect was observed in the combination of cisplatin with rutin in MCF-7 cell lines. In the in vitro metabolism studies by cytochrome P450 and Caco-2 and HepG2 cells, no metabolites of tamoxifen were detected, so it was not possible to study the interaction of natural products in its metabolism. Furthermore, when present in biological samples, tamoxifen analysis by HPLC-DAD and FTIR became difficult, probably because of its binding to BSA and also because of its hydrophobicity. Studies performed by FTIR spectroscopy revealed that the drug’s effects and its combination with natural products leads to cell death by apoptosis, through DNA fragmentation and rearrangement in the membrane lipid composition, which are the processes necessary for the formation of apoptotic bodies. The most probable interaction mechanisms are associated with the ability of natural products to interfere with drug signaling pathways, attenuating or stimulating the induction of apoptosis. In conclusion, quercetin, rutin and S. platensis digestion products can modify cellular action of chemotherapeutic drugs like tamoxifen and cisplatin in several cancer cell lines. Taking into account that the mechanism of interaction with tamoxifen may be related to its metabolism, it is necessary to develop new methods to test this hypothesis.
Currently, cancer is one of the most frequent disease in the world, with chemotherapy being the most common type of treatment. Many patients, during conventional chemotherapy, choose to use a supplemental treatment with natural products and dietary supplements claiming health benefits for cancer treatment, however there are few studies that prove their efficacy. The main aim of this study is to investigate several natural products that may affect the activity of two common chemotherapeutic drugs like tamoxifen and cisplatin. Since tamoxifen is a prodrug, it’s also necessary to study the effect of natural products on his metabolism. For this, cytotoxicity assays of two chemotherapy drugs were performed in the presence of natural compounds derived from diet: quercetin, rutin (glycosylated form of quercetin) and the products resulting from Spirulina platensis digestion. The influence of bovine serum albumin (BSA), a serum solute carrier protein, on the cytotoxicity of tamoxifen was also studied. This assays showed that the activity of tamoxifen in HeLa cell line is influenced by the flavonoids, increasing the cytotoxic effect in the presence of quercetin and decreasing in the presence of rutin. While, in MCF-7 cell line the activity of tamoxifen decreases in the presence of the products resulting from S. platensis digestion. In addition, in both cell lines, the activity of tamoxifen declines significantly in the presence of BSA, probably due to the binding of the drug to the protein. In the case of cisplatin, the cytotoxic activity decreases in the presence of quercetin and products of S. platensis digestion in HeLa cell line and decreases in the presence of quercetin, rutin and products of S. platensis digestion in MCF-7 cell line. For a better comprehension of the mechanisms underlying the interaction between drug and natural products several studies were performed – proliferation, in vitro metabolism and cellular structure changes by FTIR spectroscopy. Proliferation studies demonstrate that in HeLa cell line both drugs have an effect on the invasion mechanism used by cancer cells. The interaction of tamoxifen with rutin and S. platensis digestion products and the interaction of cisplatin with quercetin and rutin have an effect increasing adhesion and subsequent proliferation of HeLa cell line. The same effect was observed in the combination of cisplatin with rutin in MCF-7 cell lines. In the in vitro metabolism studies by cytochrome P450 and Caco-2 and HepG2 cells, no metabolites of tamoxifen were detected, so it was not possible to study the interaction of natural products in its metabolism. Furthermore, when present in biological samples, tamoxifen analysis by HPLC-DAD and FTIR became difficult, probably because of its binding to BSA and also because of its hydrophobicity. Studies performed by FTIR spectroscopy revealed that the drug’s effects and its combination with natural products leads to cell death by apoptosis, through DNA fragmentation and rearrangement in the membrane lipid composition, which are the processes necessary for the formation of apoptotic bodies. The most probable interaction mechanisms are associated with the ability of natural products to interfere with drug signaling pathways, attenuating or stimulating the induction of apoptosis. In conclusion, quercetin, rutin and S. platensis digestion products can modify cellular action of chemotherapeutic drugs like tamoxifen and cisplatin in several cancer cell lines. Taking into account that the mechanism of interaction with tamoxifen may be related to its metabolism, it is necessary to develop new methods to test this hypothesis.
Descrição
Tese de mestrado, Bioquímica (Bioquímica Médica) Universidade de Lisboa, Faculdade de Ciências, 2017
Palavras-chave
Tamoxifeno Cisplatina Quercetina Rutina Spirulina platensis Teses de mestrado - 2017