RESUMO
ABSTRACT Objective: To verify the physiological action of triiodothyronine T3 on the expression of transforming growth factor α (TGFA) mRNA in MCF7 cells by inhibition of RNA Polymerase II and the MAPK/ERK pathway Materials and methods: The cell line was treated with T3 at a physiological dose (10−9M) for 10 minutes, 1 and 4 hour (h) in the presence or absence of the inhibitors, α-amanitin (RNA polymerase II inhibitor) and PD98059 (MAPK/ERK pathway inhibitor). TGFA mRNA expression was analyzed by RT-PCR. For data analysis, we used ANOVA, complemented with the Tukey test and Student t-test, with a minimum significance of 5%. Results: T3 increases the expression of TGFA mRNA in MCF7 cells in 4 h of treatment. Inhibition of RNA polymerase II modulates the effect of T3 treatment on the expression of TGFA in MCF7 cells. Activation of the MAPK/ERK pathway is not required for T3 to affect the expression of TGFA mRNA. Conclusion: Treatment with a physiological concentration of T3 after RNA polymerase II inhibition altered the expression of TGFA. Inhibition of the MAPK/ERK pathway after T3 treatment does not interfere with the TGFA gene expression in a breast adenocarcinoma cell line.
Assuntos
Humanos , Feminino , Tri-Iodotironina/genética , Neoplasias da Mama/genética , Adenocarcinoma/genética , Regulação Neoplásica da Expressão Gênica/genética , Fator de Crescimento Transformador alfa/genética , Sistema de Sinalização das MAP Quinases/genética , Tri-Iodotironina/metabolismo , Tri-Iodotironina/farmacologia , Proto-Oncogenes/genética , Neoplasias da Mama/metabolismo , RNA Mensageiro/genética , Adenocarcinoma/metabolismo , Fator de Crescimento Transformador alfa/efeitos dos fármacos , Fator de Crescimento Transformador alfa/metabolismo , Linhagem Celular Tumoral/metabolismo , Células MCF-7/metabolismoRESUMO
OBJECTIVE: To verify the physiological action of triiodothyronine T3 on the expression of transforming growth factor α (TGFA) mRNA in MCF7 cells by inhibition of RNA Polymerase II and the MAPK/ERK pathway. MATERIALS AND METHODS: The cell line was treated with T3 at a physiological dose (10-9M) for 10 minutes, 1 and 4 hour (h) in the presence or absence of the inhibitors, α-amanitin (RNA polymerase II inhibitor) and PD98059 (MAPK/ERK pathway inhibitor). TGFA mRNA expression was analyzed by RT-PCR. For data analysis, we used ANOVA, complemented with the Tukey test and Student t-test, with a minimum significance of 5%. RESULTS: T3 increases the expression of TGFA mRNA in MCF7 cells in 4 h of treatment. Inhibition of RNA polymerase II modulates the effect of T3 treatment on the expression of TGFA in MCF7 cells. Activation of the MAPK/ERK pathway is not required for T3 to affect the expression of TGFA mRNA. CONCLUSION: Treatment with a physiological concentration of T3 after RNA polymerase II inhibition altered the expression of TGFA. Inhibition of the MAPK/ERK pathway after T3 treatment does not interfere with the TGFA gene expression in a breast adenocarcinoma cell line.
Assuntos
Adenocarcinoma/genética , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica/genética , Sistema de Sinalização das MAP Quinases/genética , Fator de Crescimento Transformador alfa/genética , Tri-Iodotironina/genética , Adenocarcinoma/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral/metabolismo , Feminino , Humanos , Células MCF-7/metabolismo , Proto-Oncogene Mas , Proto-Oncogenes/genética , RNA Mensageiro/genética , Fator de Crescimento Transformador alfa/efeitos dos fármacos , Fator de Crescimento Transformador alfa/metabolismo , Tri-Iodotironina/metabolismo , Tri-Iodotironina/farmacologiaRESUMO
Human adipose tissue-derived stem cells (hASCs) have been subjected to extensive investigation because of their self-renewal properties and potential to restore damaged tissues. In the literature, there are several protocols for differentiating hASCs into osteoblasts, but there is no report on the control of cell viability during this process. In this study, we used osteoblasts derived from hASCs of patients undergoing abdominoplasty. The cells were observed at the beginning and end of bone matrix formation, and the expression of proteins involved in this process, including alkaline phosphatase and osteocalcin, was assessed. RANKL, Osterix, Runx2, Collagen3A1, Osteopontin and BSP expression levels were analyzed using real-time PCR, in addition to a quantitative assessment of protein levels of the markers CD45, CD105, STRO-1, and Nanog, using immunofluorescence. Rhodamine (Rho123), cytochrome-c, caspase-3, P-27, cyclin D1, and autophagy cell markers were analyzed by flow cytometry to demonstrate potential cellular activity and the absence of apoptotic and tumor cell processes before and after cell differentiation. The formation of bone matrix, along with calcium nodules, was observed after 16 days of osteoinduction. The gene expression levels of RANKL, Osterix, Runx2, Collagen3A1, Osteopontin, BSP and alkaline phosphatase activity were also elevated after 16 days of osteoinduction, whereas the level of osteocalcin was higher after 21 days of osteoinduction. Our data also showed that the cells had a high mitochondrial membrane potential and a low expression of apoptotic and tumor markers, both before and after differentiation. Cells were viable after the different phases of differentiation. This proposed methodology, using markers to evaluate cell viability, is therefore successful in assessing different phases of stem cell isolation and differentiation.
Assuntos
Tecido Adiposo/citologia , Sobrevivência Celular , Osteoblastos/citologia , Células-Tronco/citologia , Fosfatase Alcalina/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Células Cultivadas , Colágeno Tipo III/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica , Humanos , Potencial da Membrana Mitocondrial , Microscopia de Fluorescência , Modelos Biológicos , Osteoblastos/metabolismo , Osteopontina/metabolismo , Ligante RANK/metabolismo , Vimentina/metabolismoRESUMO
ABSTRACT Objective The current study was aimed at analyzing sarcoplasmic reticulum Ca2+ ATPase (Serca2) and ryanodine receptor type 2 (Ryr2) gene expression in rats subjected to surgery that induced HF and were subsequently treated with T4 using physiological doses. Materials and methods HF was induced in 18 male Wistar rats by clipping the ascending thoracic aorta to generate aortic stenosis (HFS group), while the control group (9-sham) underwent thoracotomy. After 21 weeks, the HFS group was subdivided into two subgroups. One group (9 Wistar rats) with HF received 1.0 µg of T4/100 g of body weight for five consecutive days (HFS/T4); the other group (9 Wistar rats) received isotonic saline solution (HFS/S). The animals were sacrificed after this treatment and examined for signs of HF. Samples from the left ventricles of these animals were analyzed by RT-qPCR for the expression of Serca2 and Ryr2 genes. Results Rats with HF developed euthyroid sick syndrome (ESS) and treatment with T4 restored the T3 values to the Sham level and increased Serca2 and Ryr2 gene expression, thereby demonstrating a possible benefit of T4 treatment for heart function in ESS associated with HF. Conclusion The T4 treatment can potentially normalize the levels of T3 as well elevated Serca2 and Ryr2 gene expression in the myocardium in heart failure rats with euthyroid sick syndrome.
Assuntos
Animais , Masculino , Tiroxina/administração & dosagem , Síndromes do Eutireóideo Doente/tratamento farmacológico , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Estenose da Valva Aórtica/complicações , Tiroxina/uso terapêutico , Tri-Iodotironina/efeitos dos fármacos , Síndromes do Eutireóideo Doente/complicações , Síndromes do Eutireóideo Doente/genética , RNA Mensageiro/metabolismo , Expressão Gênica/efeitos dos fármacos , Ratos Wistar , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Modelos Animais , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Insuficiência Cardíaca/complicaçõesRESUMO
OBJECTIVE: The current study was aimed at analyzing sarcoplasmic reticulum Ca2+ ATPase (Serca2) and ryanodine receptor type 2 (Ryr2) gene expression in rats subjected to surgery that induced HF and were subsequently treated with T4 using physiological doses. MATERIALS AND METHODS: HF was induced in 18 male Wistar rats by clipping the ascending thoracic aorta to generate aortic stenosis (HFS group), while the control group (9-sham) underwent thoracotomy. After 21 weeks, the HFS group was subdivided into two subgroups. One group (9 Wistar rats) with HF received 1.0 µg of T4/100 g of body weight for five consecutive days (HFS/T4); the other group (9 Wistar rats) received isotonic saline solution (HFS/S). The animals were sacrificed after this treatment and examined for signs of HF. Samples from the left ventricles of these animals were analyzed by RT-qPCR for the expression of Serca2 and Ryr2 genes. RESULTS: Rats with HF developed euthyroid sick syndrome (ESS) and treatment with T4 restored the T3 values to the Sham level and increased Serca2 and Ryr2 gene expression, thereby demonstrating a possible benefit of T4 treatment for heart function in ESS associated with HF. CONCLUSION: The T4 treatment can potentially normalize the levels of T3 as well elevated Serca2 and Ryr2 gene expression in the myocardium in heart failure rats with euthyroid sick syndrome.