Estudo da ação de derivados semi-sintéticos de curcumina em linhagens celulares de câncer humano
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Data
2016-03-01
Autores
Oliveira, Ana Beatriz Bortolozo de [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Os Papilomavírus humanos (HPVs) são vírus de DNA divididos em dois grupos. O de baixo risco, associado a lesões benignas, e o de alto risco, associado a diversos tipos de câncer. As oncoproteínas E6 e E7 são as principais proteínas envolvidas no processo de carcinogênese decorrente da infecção por esses vírus, de forma que compostos naturais têm sido utilizados na busca por drogas que inibam a expressão viral. Paralelamente, devido aos diversos efeitos colaterais e alto índice de reincidência dos tratamentos tradicionais contra o câncer, a procura por agentes antitumorais é intensa. Nesse contexto, diversas pesquisas estão sendo realizadas afim de explorar as inúmeras propriedades da curcumina, uma substância que apresenta atividade antiinflamatória, imuno-modulatória, antioxidante, anti-angiogênica, quimioprotetora, antiproliferativa, antitumoral e antimicrobiana. Assim, uma estratégia promissora utilizada é a busca por análogos de curcumina que consigam aumentar a eficácia terapêutica desse fitoterápico. Dessa forma, o objetivo desse trabalho é analisar in vitro a ação de diferentes derivados semi-sintéticos de curcumina (AC1, AC2, AC7, AC10 E AC13) sobre a expressão dos oncogenes virais E6 e E7 em linhagem de carcinoma cervical HPV-16 positiva (CasKi) e a atividade antitumoral do composto AC13 em diferentes linhagens tumorais (CasKi, HeLa, MDA-MB- 231, MCF-7 e 786-O). Para isso, após a incubação com os compostos em diferentes concentrações, a viabilidade das células foi analisada por ensaio MTT e uma linhagem imortal transformada de queratinócito humano (HaCaT) foi utilizada como controle para análise da citotoxicidade. Posteriormente, a avaliação da expressão do RNAm de E6 e E7 foi realizada por PCR em tempo real, a determinação da expressão da proteína p53 por Western Blotting e a detecção da atividade das caspases 3 e 7 foi analisada por meio de ensaio luminescente de apoptose. Embora nenhum composto tenha inibido a expressão de E6 e E7 na linhagem CasKi, as análises de citotoxicidade do composto AC13 sugerem que ele apresente vantagens em relação à curcumina nessa linhagem celular, pois foi menos citotóxico do que a curcumina para as células não tumorais (HaCaT) e causou queda de viabilidade um pouco maior para a linhagem CasKi do que para a HaCaT nas concentrações de 50, 75 e 100 μM. Além disso, o AC13 induz aumento da atividade das caspases, o que indica que as células são levadas a morte por apoptose. Dessa forma, os dados obtidos sugerem que o derivado de curcumina AC13 apresenta interessante atividade antitumoral nas células CasKi.
High risk Human Papilomavírus (HPVs) are DNA vírus divided in two groups. Low risk, associated with benign lesions, and high risk, associated with several types of cancer. Oncoproteins HPV E6 and E7 are the main proteins involved in the carcinogenesis process due to infection by this virus, so that natural coumponds have been used in the search for drugs that inhibit viral expression. At the same time, due to various side effects and high relapse rate of traditional cancer treatments, the search for antitumor agents is intense. In this context, several studies are being conducted in order to explore the numerous properties of curcumin, a substance that has anti-inflammatory, immune-modulatory, anti-oxidant, anti-angiogenic, chemoprotective, anti-proliferative, anti-tumor and anti-microbial properties. Thus, a promising strategy used is the search for curcumin analogues which are able to increase the therapeutic efficacy of phytotherapy. Thus, the aim of this study is to analyze the action of different semi-synthetic derivatives of curcumin (AC1, AC2, AC7, AC10 and AC13) on the E6 and E7 viral oncogenes expression in positive HPV-16 cervical carcinoma cell line (CasKi) and the antitumor activity of AC13 compound in different tumor cell lines (CasKi, HeLa, MDA 231, MCF-7 and 786-O). For this, after incubation with the compounds at different concentrations, cell viability was analyzed by MTT assay and a spontaneously transformed immortal keratinocyte cell line (HaCaT) was used as a control to analyze the cytotoxicity. Subsequently, E6 and E7 mRNA expression evaluation was performed by real-time PCR, p53 protein expression determination was analyzed by Western Blotting, and detecting the caspases 3 and 7 activity was analyzed using the luminescent assay of apoptosis. Although no compound has inhibited E6 and E7 expression in the CasKi cell line, AC13 cytotoxicity analyzes suggest that it has advantages in relation to curcumin in this cell line, because it was more cytotoxic than curcumin to no tumoral cells (HaCaT) and caused viability decrease less pronunced for CasKi line than for HaCaT at 50, 75 e 100 μM. Furthermore, AC13 induces increased caspases activity, suggesting cell death by apoptosis. Thus, our datas suggest that AC13 derivative of curcumin presents interesting antitumor activity in CasKi cells.
High risk Human Papilomavírus (HPVs) are DNA vírus divided in two groups. Low risk, associated with benign lesions, and high risk, associated with several types of cancer. Oncoproteins HPV E6 and E7 are the main proteins involved in the carcinogenesis process due to infection by this virus, so that natural coumponds have been used in the search for drugs that inhibit viral expression. At the same time, due to various side effects and high relapse rate of traditional cancer treatments, the search for antitumor agents is intense. In this context, several studies are being conducted in order to explore the numerous properties of curcumin, a substance that has anti-inflammatory, immune-modulatory, anti-oxidant, anti-angiogenic, chemoprotective, anti-proliferative, anti-tumor and anti-microbial properties. Thus, a promising strategy used is the search for curcumin analogues which are able to increase the therapeutic efficacy of phytotherapy. Thus, the aim of this study is to analyze the action of different semi-synthetic derivatives of curcumin (AC1, AC2, AC7, AC10 and AC13) on the E6 and E7 viral oncogenes expression in positive HPV-16 cervical carcinoma cell line (CasKi) and the antitumor activity of AC13 compound in different tumor cell lines (CasKi, HeLa, MDA 231, MCF-7 and 786-O). For this, after incubation with the compounds at different concentrations, cell viability was analyzed by MTT assay and a spontaneously transformed immortal keratinocyte cell line (HaCaT) was used as a control to analyze the cytotoxicity. Subsequently, E6 and E7 mRNA expression evaluation was performed by real-time PCR, p53 protein expression determination was analyzed by Western Blotting, and detecting the caspases 3 and 7 activity was analyzed using the luminescent assay of apoptosis. Although no compound has inhibited E6 and E7 expression in the CasKi cell line, AC13 cytotoxicity analyzes suggest that it has advantages in relation to curcumin in this cell line, because it was more cytotoxic than curcumin to no tumoral cells (HaCaT) and caused viability decrease less pronunced for CasKi line than for HaCaT at 50, 75 e 100 μM. Furthermore, AC13 induces increased caspases activity, suggesting cell death by apoptosis. Thus, our datas suggest that AC13 derivative of curcumin presents interesting antitumor activity in CasKi cells.
Descrição
Palavras-chave
HPV, Câncer, Curcumina, Derivado semi-sintético, Antitumoral, Cancer, Curcumin, Semi-synthetic derivatives, Antitumoral