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. |
|---|---|
| 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 |
| _version_ | 1867438314091446272 |
|---|---|
| author | Vale, Diogo Miguel Reis do |
| author_facet | Vale, Diogo Miguel Reis do |
| author_role | author |
| contributor_name_str_mv | Pereira-Wilson, Cristina RepositóriUM - Universidade do Minho |
| country_str | PT |
| creators_json_txt | [{\"Person.name\":\"Vale, Diogo Miguel Reis do\"}] |
| datacite.contributors.contributor.contributorName.fl_str_mv | Pereira-Wilson, Cristina RepositóriUM - Universidade do Minho |
| datacite.creators.creator.creatorName.fl_str_mv | Vale, Diogo Miguel Reis do |
| datacite.date.Accepted.fl_str_mv | 2018-01-01T00:00:00Z |
| datacite.date.available.fl_str_mv | 2022-01-01T07:00:32Z |
| datacite.date.embargoed.fl_str_mv | 2022-01-01T07:00:32Z |
| datacite.rights.fl_str_mv | http://purl.org/coar/access_right/c_abf2 |
| datacite.subjects.subject.fl_str_mv | Ciências Naturais::Ciências Biológicas |
| datacite.titles.title.fl_str_mv | The use of the comet assay to characterize the effects of dietary constituents on the prevention and treatment of colon cancer |
| dc.contributor.none.fl_str_mv | Pereira-Wilson, Cristina RepositóriUM - Universidade do Minho |
| dc.creator.none.fl_str_mv | Vale, Diogo Miguel Reis do |
| dc.date.Accepted.fl_str_mv | 2018-01-01T00:00:00Z |
| dc.date.available.fl_str_mv | 2022-01-01T07:00:32Z |
| dc.date.embargoed.fl_str_mv | 2022-01-01T07:00:32Z |
| dc.description.none.fl_str_mv | O cancro colorretal (CCR) é uma doença que afeta vários homens e mulheres em todo o mundo. Embora a prevenção e os tratamentos atuais tenham diminuído a incidência do CCR e aumentado a esperança média de vida dos pacientes, têm sido registado um aumento do número de casos de CRC em algumas regiões da América do Sul e leste da Europa. Um dos vários fatores que está relacionado com o aparecimento do CRC é a dieta, uma vez que vários estudos têm estabelecido uma relação positiva entre o consumo regular de alimentos ricos em gorduras ou processados e um maior risco de desenvolvimento do CRC. Por outro lado, uma dieta equilibrada e rica em alimentos vegetais, como as leguminosas (soja ou feijão preto), pode prevenir o aparecimento de vários tipos de cancro, incluindo o cancro colorretal. As isoflavonas, como a genisteína e a daidzeína, são alguns dos compostos que estão naturalmente presentes na soja e em produtos à base de soja e que têm efeitos benéficos no combate ao desenvolvimento e progressão de tumores. Neste projeto, foi avaliado, pelo ensaio cometa, o efeito da combinação da genisteína e daidzeína com as drogas quimioterápicas 5-Fluorouracil e oxaliplatina na indução de danos no DNA nas linhas celulares de cancro colorretal RKO e HCT116. As células foram expostas a tratamentos combinados por um período de dois a quatro dias com doses IC25 dos compostos, obtidas previamente pela medição dos seus efeitos citotóxicos com a ajuda do ensaio MTT. Foi também analisada a indução de apoptose pelo ensaio de condensação nuclear e realizada uma reação em cadeia de polimerase em tempo real (qPCR) para avaliar se este tipo de terapia pode reverter, nas RKO, o silenciamento epigenético do gene MutL homolog 1 (MLH1) e aumentar a atividade do p16, responsáveis, respetivamente, pela reparação de danos no DNA e pelo controlo do ciclo celular. A 5-Azacitidina foi também utilizada neste estudo como um controlo positivo desmetilante. Neste trabalho, foi observado que o 5-Fluorouracil induz quebras no DNA das células RKO e que a co-incubação com as isoflavonas diminuiu significativamente a viabilidade celular. No entanto, esta diminuição não se refletiu num aumento da apoptose ou num aumento do número de quebras no DNA. O teste qPCR revelou que nenhum dos tratamentos foi capaz de reativar a expressão do MLH1 ou aumentar os níveis de p16, exceto as células que foram tratadas com a 5-Azacitidina. Este estudo demostrou que a combinação de drogas quimioterápicas com a genisteína e a daidzeína pode aumentar a eficácia do tratamento do CCR e que o ensaio cometa é uma ferramenta promissora na análise dos efeitos deste tipo de terapia em células de cancro colorretal. |
| dc.format.none.fl_str_mv | application/pdf |
| dc.identifier.none.fl_str_mv | https://hdl.handle.net/1822/65040 |
| dc.language.none.fl_str_mv | eng |
| dc.rights.none.fl_str_mv | http://purl.org/coar/access_right/c_abf2 |
| dc.subject.none.fl_str_mv | Ciências Naturais::Ciências Biológicas |
| dc.title.fl_str_mv | The use of the comet assay to characterize the effects of dietary constituents on the prevention and treatment of colon cancer |
| dc.type.none.fl_str_mv | http://purl.org/coar/resource_type/c_bdcc |
| description | 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. |
| dirty | 0 |
| eu_rights_str_mv | openAccess |
| format | masterThesis |
| fulltext.url.fl_str_mv | https://repositorium.uminho.pt/bitstreams/2450a575-125c-4d7a-8a0a-101c21ab17ff/download |
| id | rum_df3d17bb37cf2fdc3eb2bb4a7afbe710 |
| identifier.url.fl_str_mv | https://hdl.handle.net/1822/65040 |
| instacron_str | repositorium |
| institution | Universidade do Minho |
| instname_str | Universidade do Minho |
| language | eng |
| network_acronym_str | rum |
| network_name_str | RepositóriUM - Universidade do Minho |
| oai_identifier_str | oai:repositorium.uminho.pt:1822/65040 |
| organization_str_mv | urn:organizationAcronym:repositorium |
| person_str_mv | Vale, Diogo Miguel Reis do |
| publishDate | 2018 |
| reponame_str | RepositóriUM - Universidade do Minho |
| repository_id_str | urn:repositoryAcronym:rum |
| service_str_mv | urn:repositoryAcronym:rum |
| spelling | engengColorectal 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.porO cancro colorretal (CCR) é uma doença que afeta vários homens e mulheres em todo o mundo. Embora a prevenção e os tratamentos atuais tenham diminuído a incidência do CCR e aumentado a esperança média de vida dos pacientes, têm sido registado um aumento do número de casos de CRC em algumas regiões da América do Sul e leste da Europa. Um dos vários fatores que está relacionado com o aparecimento do CRC é a dieta, uma vez que vários estudos têm estabelecido uma relação positiva entre o consumo regular de alimentos ricos em gorduras ou processados e um maior risco de desenvolvimento do CRC. Por outro lado, uma dieta equilibrada e rica em alimentos vegetais, como as leguminosas (soja ou feijão preto), pode prevenir o aparecimento de vários tipos de cancro, incluindo o cancro colorretal. As isoflavonas, como a genisteína e a daidzeína, são alguns dos compostos que estão naturalmente presentes na soja e em produtos à base de soja e que têm efeitos benéficos no combate ao desenvolvimento e progressão de tumores. Neste projeto, foi avaliado, pelo ensaio cometa, o efeito da combinação da genisteína e daidzeína com as drogas quimioterápicas 5-Fluorouracil e oxaliplatina na indução de danos no DNA nas linhas celulares de cancro colorretal RKO e HCT116. As células foram expostas a tratamentos combinados por um período de dois a quatro dias com doses IC25 dos compostos, obtidas previamente pela medição dos seus efeitos citotóxicos com a ajuda do ensaio MTT. Foi também analisada a indução de apoptose pelo ensaio de condensação nuclear e realizada uma reação em cadeia de polimerase em tempo real (qPCR) para avaliar se este tipo de terapia pode reverter, nas RKO, o silenciamento epigenético do gene MutL homolog 1 (MLH1) e aumentar a atividade do p16, responsáveis, respetivamente, pela reparação de danos no DNA e pelo controlo do ciclo celular. A 5-Azacitidina foi também utilizada neste estudo como um controlo positivo desmetilante. Neste trabalho, foi observado que o 5-Fluorouracil induz quebras no DNA das células RKO e que a co-incubação com as isoflavonas diminuiu significativamente a viabilidade celular. No entanto, esta diminuição não se refletiu num aumento da apoptose ou num aumento do número de quebras no DNA. O teste qPCR revelou que nenhum dos tratamentos foi capaz de reativar a expressão do MLH1 ou aumentar os níveis de p16, exceto as células que foram tratadas com a 5-Azacitidina. Este estudo demostrou que a combinação de drogas quimioterápicas com a genisteína e a daidzeína pode aumentar a eficácia do tratamento do CCR e que o ensaio cometa é uma ferramenta promissora na análise dos efeitos deste tipo de terapia em células de cancro colorretal.application/pdfengThe use of the comet assay to characterize the effects of dietary constituents on the prevention and treatment of colon cancerVale, Diogo Miguel Reis doPereira-Wilson, CristinaHostingInstitutionOrganizationalRepositóriUM - Universidade do Minhoe-mailmailto:repositorium@usdb.uminho.ptrepositorium@usdb.uminho.ptTID2022141412022-01-01T07:00:32Z201820182018-01-01T00:00:00ZHandlehttps://hdl.handle.net/1822/65040http://purl.org/coar/access_right/c_abf2open accesshttp://www.oecd.org/science/inno/38235147.pdfFields of Science and Technology (FOS)Ciências Naturais::Ciências Biológicas3407494 bytesliteraturehttp://purl.org/coar/resource_type/c_bdccmaster thesishttp://purl.org/coar/access_right/c_abf2application/pdffulltexthttps://repositorium.uminho.pt/bitstreams/2450a575-125c-4d7a-8a0a-101c21ab17ff/download |
| spellingShingle | The use of the comet assay to characterize the effects of dietary constituents on the prevention and treatment of colon cancer Vale, Diogo Miguel Reis do Ciências Naturais::Ciências Biológicas |
| status | SINGLETON |
| subject.other.fl_str_mv | Ciências Naturais::Ciências Biológicas |
| title | The use of the comet assay to characterize the effects of dietary constituents on the prevention and treatment of colon cancer |
| title_full | The use of the comet assay to characterize the effects of dietary constituents on the prevention and treatment of colon cancer |
| title_fullStr | The use of the comet assay to characterize the effects of dietary constituents on the prevention and treatment of colon cancer |
| title_full_unstemmed | The use of the comet assay to characterize the effects of dietary constituents on the prevention and treatment of colon cancer |
| title_short | The use of the comet assay to characterize the effects of dietary constituents on the prevention and treatment of colon cancer |
| title_sort | The use of the comet assay to characterize the effects of dietary constituents on the prevention and treatment of colon cancer |
| topic | Ciências Naturais::Ciências Biológicas |
| topic_facet | Ciências Naturais::Ciências Biológicas |
| url | https://hdl.handle.net/1822/65040 |
| visible | 1 |