Avaliação do efeito do fármaco parecoxib na linha celular MG-63
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2019-03-21
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Inúmeras neoplasias, entre elas o osteossarcoma, apresentam uma sobre expressão da cicloxigenase-2 (COX-2) e, consequentemente de prostaglandinas, associadas a um pior prognóstico. Como tal, fármacos que inibam a COX-2 podem representar uma opção na terapia do cancro. Este estudo teve como principal objetivo avaliar o efeito in vitro do parecoxib (fármaco anti-inflamatório não esteroide, inibidor seletivo da COX-2) na linha celular de osteossarcoma humano MG-63. Para alcançar o objetivo proposto, realizou-se a validação da linha celular por técnicas de citogenética clássica. Por hibridação fluorescente in situ (FISH) foi estudado a amplificação do gene HER2 e a translocação do gene ALK. Posteriormente, as células foram expostas a diferentes concentrações de parecoxib (0, 50, 100, 150, 200, 250, 300, 350, 400, 800, 1200 e 1600 µM), durante um intervalo de 48 h. A viabilidade celular foi avaliada através do ensaio do brometo de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio (MTT) e permitiu a obtenção do valor do IC50 (inhibitory maximal concentration for half of the population) (163 μM). Este valor, bem como a concentração mais elevada testada (1600 μM), foram utilizados nos ensaios subsequentes. A proliferação celular, a integridade membranar, a avaliação do ciclo celular, a apoptose, a autofagia, os danos no DNA e as alterações morfológicas celulares foram também avaliadas, por imunocitoquímica (Ki-67), deteção da LDH, citometria de fluxo (iodeto de propídio), terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL), coloração com o laranja de acridina, ensaio do cometa alcalino e coloração de Leishman, respetivamente. Foram identificadas várias alterações cromossómicas na linha celular MG-63, as quais se encontram de acordo com o descrito na literatura. Através da técnica de FISH, não se observaram alterações nos genes ALK/HER2. Comparativamente ao grupo de células não tratadas, pelo ensaio do MTT e da imunocitoquímica, verificou-se respetivamente, que o parecoxib originou uma diminuição quer da viabilidade, quer da proliferação celular, de forma dependente da concentração. A deteção da lactato desidrogenase (LDH) permitiu observar um aumento da libertação desta enzima, semelhante para ambas as concentrações testadas. Por citometria de fluxo, verificou-se uma diminuição das células que se encontram em G0/G1 e uma paragem do ciclo celular nas fases S e G2/M, bem como um aumento da fração sub-G0/G1. O TUNEL revelou um aumento da apoptose nas células tratadas. A coloração com o laranja de acridina permitiu observar um aumento da presença de vacúolos autofágicos, após exposição ao parecoxib. Através do ensaio do cometa alcalino foi possível observar um aumento dos danos no DNA, dependente da concentração. Pela coloração de Leishman, constatou-se uma maior incidência de corpos apoptóticos e citoplasma vacuolizado, proporcional à concentração testada. O parecoxib demonstrou ter efeito nas células de osteossarcoma humano MG-63, levando à diminuição da viabilidade/proliferação celular, à perda da integridade membranar, à paragem do ciclo celular e ao aumento da apoptose/autofagia. Estes resultados sugerem que o parecoxib pode representar um bom candidato para estudos in vivo.
Different types of tumors, namely osteosarcoma, present an overexpression of COX-2 and therefore of prostaglandins, which are associated with a worse prognosis. Consequently, drugs that inhibit COX-2 may represent an option in cancer therapy. The aim of this study was to evaluate the effect of parecoxib (nonsteroidal antiinflammatory drug, COX-2 selective inhibitor), in vitro, in the human osteosarcoma cell line MG-63. Cell line validation was performed using classical cytogenetic techniques. The HER2 gene amplification and ALK gene translocation were studied by fluorescent in situ hybridization (FISH) technique. Cells were exposed to several concentrations of parecoxib (0, 50, 100, 150, 200, 250, 300, 350, 400, 800, 1200 e 1600 µM), for a 48h period. Cell viability was studied through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT). The inhibitory maximal concentration for half of the population - IC50 - was determined by this method (163 µM). The IC50 concentration, as well as the highest concentration tested (1600 µM) were applied for the rest of the assays. Cell proliferation, membrane integrity, cell cycle evaluation, apoptosis, autophagy, DNA damage and morphologic alterations were also evaluated in this study, through immunocytochemistry (Ki67), LDH detection, flow cytometry (propidium iodide), terminal deoxynucleotidyltransferasemediated dUTP nick end labeling (TUNEL), acridine orange staining, alkaline comet assay and Leishman staining, respectively. Were identified several chromosomic alterations in this cell line, which are in accordance with those described in the literature. There weren’t detected any alterations in ALK and HER2 genes, by FISH technique. When compared with non-treated cells, parecoxib led to a demarked decrease of cell viability and proliferation, in a concentration-dependent manner, by means of MTT assay and immunocytochemistry, respectively. Lactate dehydrogenase (LDH) detection showed a higher enzyme release, similar for both concentrations. In flow cytometry was observed a decrease of cells found in G0/G1-phase and an arrest of the cell cycle at S-phase and G2/M-phase, as well as a growth of the sub-G0/G1-fraction. TUNEL assay revealed a slight increment of apoptosis in treated cells. Acridine orange staining showed an increase of autophagic vacuoles, after parecoxib exposure. The DNA damage, assessed by the comet assay, was increased, in treated cells, in a concentration-dependent manner. With Leishman staining a higher incidence of apoptotic bodies and vacuolated cytoplasm were observed. Parecoxib showed effect in the human osteosarcoma MG-63 cells, through the decrease in cell viability/proliferation, loss of membrane integrity, arrest of cell cycle, and increase of apoptosis/autophagy. These results further suggested that parecoxib might be a potential candidate for in vivo studies.
Different types of tumors, namely osteosarcoma, present an overexpression of COX-2 and therefore of prostaglandins, which are associated with a worse prognosis. Consequently, drugs that inhibit COX-2 may represent an option in cancer therapy. The aim of this study was to evaluate the effect of parecoxib (nonsteroidal antiinflammatory drug, COX-2 selective inhibitor), in vitro, in the human osteosarcoma cell line MG-63. Cell line validation was performed using classical cytogenetic techniques. The HER2 gene amplification and ALK gene translocation were studied by fluorescent in situ hybridization (FISH) technique. Cells were exposed to several concentrations of parecoxib (0, 50, 100, 150, 200, 250, 300, 350, 400, 800, 1200 e 1600 µM), for a 48h period. Cell viability was studied through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT). The inhibitory maximal concentration for half of the population - IC50 - was determined by this method (163 µM). The IC50 concentration, as well as the highest concentration tested (1600 µM) were applied for the rest of the assays. Cell proliferation, membrane integrity, cell cycle evaluation, apoptosis, autophagy, DNA damage and morphologic alterations were also evaluated in this study, through immunocytochemistry (Ki67), LDH detection, flow cytometry (propidium iodide), terminal deoxynucleotidyltransferasemediated dUTP nick end labeling (TUNEL), acridine orange staining, alkaline comet assay and Leishman staining, respectively. Were identified several chromosomic alterations in this cell line, which are in accordance with those described in the literature. There weren’t detected any alterations in ALK and HER2 genes, by FISH technique. When compared with non-treated cells, parecoxib led to a demarked decrease of cell viability and proliferation, in a concentration-dependent manner, by means of MTT assay and immunocytochemistry, respectively. Lactate dehydrogenase (LDH) detection showed a higher enzyme release, similar for both concentrations. In flow cytometry was observed a decrease of cells found in G0/G1-phase and an arrest of the cell cycle at S-phase and G2/M-phase, as well as a growth of the sub-G0/G1-fraction. TUNEL assay revealed a slight increment of apoptosis in treated cells. Acridine orange staining showed an increase of autophagic vacuoles, after parecoxib exposure. The DNA damage, assessed by the comet assay, was increased, in treated cells, in a concentration-dependent manner. With Leishman staining a higher incidence of apoptotic bodies and vacuolated cytoplasm were observed. Parecoxib showed effect in the human osteosarcoma MG-63 cells, through the decrease in cell viability/proliferation, loss of membrane integrity, arrest of cell cycle, and increase of apoptosis/autophagy. These results further suggested that parecoxib might be a potential candidate for in vivo studies.
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Dissertação de Mestrado em Bioquímica
Palavras-chave
in vitro , AINES , osteossarcoma