Publicação
The use of the comet assay to characterize the effects of dietary constituents on the prevention and treatment of colon cancer
| Resumo: | Colorectal cancer (CRC) is a serious disease that affects both man and women in several countries in the world. Although prevention and current treatments (chemotherapy and radiotherapy) have been able to decrease CRC incidence and extend patient’s life expectancy, there are some regions, especially in South America and Eastern Europe, where CRC cases have been increasing in the last years. One of the several factors that is related with the CRC development is diet. The regular consumption of fat or processed foods, typical of developed countries, is associated with an increased risk of CRC. On the other hand, studies have shown that a balanced diet based on vegetable foods such as legumes (soybean or black bean) can prevent the appearance of several types of cancer, including colorectal. Isoflavones like genistein and daidzein are some of the compounds that are present in soybean and soy-based products and have beneficial effects against tumor development and progression. In this project, it was evaluated the effect of genistein and daidzein combination with the chemotherapeutic drugs 5-Fluorouracil and oxaliplatin on the induction of DNA damage in RKO and HCT116 colorectal cancer cell lines by comet assay. Cells were exposed to combined treatments for a period of two to four days with IC25 doses of the compounds, previously obtained by measuring cytotoxic effects with the help of the MTT assay. It was analyzed the percentage of DNA strand breaks produced in each regimen and apoptosis induction by the nuclear condensation assay. It was also performed a real time polymerase chain reaction (qPCR) to evaluate if this type of therapy could revert, in RKO, the epigenetic silencing of the gene MutL homolog 1 (MLH1) and have effects on p16, responsible for DNA mismatch repair and the control of cell cycle, respectively. 5-Azacytidine was also used in this study as a demethylating positive control. We observed that 5-Fluorouracil produces DNA strand breaks in RKO cells and the co-incubation with genistein or daidzein significantly decreased cell viability. This decreased viability was, however, not reflected in increased apoptosis or DNA strand breaks. The qPCR test revealed that none of the treatments were able to reactivate the expression of MLH1 or increased p16 levels, with the exception of the ones that were treated with 5-Azacytidine (positive control). This study showed that chemotherapeutic drugs combined with genistein and daidzein can increase the efficiency of CRC treatment. Besides, comet assay is also a promising tool to analyze the effects of this type of therapy in colorectal cancer cells, although the isoflavone effects could not be attributed to increased apoptosis or reversal of MLH1 expression. |
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| Autores principais: | Vale, Diogo Miguel Reis do |
| Assunto: | Ciências Naturais::Ciências Biológicas |
| Ano: | 2018 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Colorectal cancer (CRC) is a serious disease that affects both man and women in several countries in the world. Although prevention and current treatments (chemotherapy and radiotherapy) have been able to decrease CRC incidence and extend patient’s life expectancy, there are some regions, especially in South America and Eastern Europe, where CRC cases have been increasing in the last years. One of the several factors that is related with the CRC development is diet. The regular consumption of fat or processed foods, typical of developed countries, is associated with an increased risk of CRC. On the other hand, studies have shown that a balanced diet based on vegetable foods such as legumes (soybean or black bean) can prevent the appearance of several types of cancer, including colorectal. Isoflavones like genistein and daidzein are some of the compounds that are present in soybean and soy-based products and have beneficial effects against tumor development and progression. In this project, it was evaluated the effect of genistein and daidzein combination with the chemotherapeutic drugs 5-Fluorouracil and oxaliplatin on the induction of DNA damage in RKO and HCT116 colorectal cancer cell lines by comet assay. Cells were exposed to combined treatments for a period of two to four days with IC25 doses of the compounds, previously obtained by measuring cytotoxic effects with the help of the MTT assay. It was analyzed the percentage of DNA strand breaks produced in each regimen and apoptosis induction by the nuclear condensation assay. It was also performed a real time polymerase chain reaction (qPCR) to evaluate if this type of therapy could revert, in RKO, the epigenetic silencing of the gene MutL homolog 1 (MLH1) and have effects on p16, responsible for DNA mismatch repair and the control of cell cycle, respectively. 5-Azacytidine was also used in this study as a demethylating positive control. We observed that 5-Fluorouracil produces DNA strand breaks in RKO cells and the co-incubation with genistein or daidzein significantly decreased cell viability. This decreased viability was, however, not reflected in increased apoptosis or DNA strand breaks. The qPCR test revealed that none of the treatments were able to reactivate the expression of MLH1 or increased p16 levels, with the exception of the ones that were treated with 5-Azacytidine (positive control). This study showed that chemotherapeutic drugs combined with genistein and daidzein can increase the efficiency of CRC treatment. Besides, comet assay is also a promising tool to analyze the effects of this type of therapy in colorectal cancer cells, although the isoflavone effects could not be attributed to increased apoptosis or reversal of MLH1 expression. |
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