Publicação
Functional characterization of mitochondrial suspensions by cytofluorimetric techniques towards the screening of mitochondrial target drugs
| Resumo: | Besides its role in respiration, mitochondria control other key metabolic pathways, cell proliferation and regulated cell death, with a crucial role in the regulation of the intrinsic apoptotic pathway. Recent studies have focused on the screen for anti-cancer agents that induce apoptosis through permeabilization of the mitochondrial outer membrane, leading to the cytosolic release of pro-apoptotic proteins and to the activation of the caspase cascade and consequently to cell dismantling. In the present study, staining protocols were developed and optimized aiming at the structural and functional characterization of isolated mitochondrial populations from yeast cells and rat liver by flow cytometry. Mitotracker Green and Nonyl Acridine Orange were used to measure changes in mitochondrial mass, 3,3'- Dihexyloxacarbocyanine Iodide and Mitotracker Red CMXROS to monitor the electrical potential of the inner membrane (Δψm), Mitotracker Red CM-H2XROS and Dihydroethidium to evaluate the accumulation of ROS, and 2',7'-Bis-(2-Carboxyethyl)- 5-(and-6)-Carboxyfluorescein and Acetoxymethyl Ester (BCECF-AM) to measure the pH of the mitochondrial matrix. Subsequent flow cytometry studies were designed to evaluate the effect in mitochondria function of two well-known apoptosis inducers in both mammalian cell lines and yeast cells, namely bovine lactoferrin (bLf) and acetate, and to understand whether or not they act directly on mitochondria independently of upstream cellular pathways. The results showed that both bLf and acetate promoted changes in mitochondrial mass, matrix mitochondrial pH, Δψm and ROS levels of rat liver mitochondria. Moreover, the assessment of the activity of the complexes of electron transport chain (ETC) showed that bLf and acetate affect their activity and trigger mitochondrial swelling. These results are particularly interesting since the two compounds appear to induce in isolated mitochondria mainly the same alterations as those occurring in whole cells, and which have been proposed to be involved in the induction of cell death. Altogether, this study show that flow cytometry is a valuable tool to study key mitochondrial functional parameters and to screen for mitochondriatargeted drugs, including the anticancer agents, such as acetate and bLf. |
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| Autores principais: | Loureiro, Luís Carlos Sá |
| Assunto: | Ciências Naturais::Ciências Biológicas |
| Ano: | 2015 |
| 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: | Besides its role in respiration, mitochondria control other key metabolic pathways, cell proliferation and regulated cell death, with a crucial role in the regulation of the intrinsic apoptotic pathway. Recent studies have focused on the screen for anti-cancer agents that induce apoptosis through permeabilization of the mitochondrial outer membrane, leading to the cytosolic release of pro-apoptotic proteins and to the activation of the caspase cascade and consequently to cell dismantling. In the present study, staining protocols were developed and optimized aiming at the structural and functional characterization of isolated mitochondrial populations from yeast cells and rat liver by flow cytometry. Mitotracker Green and Nonyl Acridine Orange were used to measure changes in mitochondrial mass, 3,3'- Dihexyloxacarbocyanine Iodide and Mitotracker Red CMXROS to monitor the electrical potential of the inner membrane (Δψm), Mitotracker Red CM-H2XROS and Dihydroethidium to evaluate the accumulation of ROS, and 2',7'-Bis-(2-Carboxyethyl)- 5-(and-6)-Carboxyfluorescein and Acetoxymethyl Ester (BCECF-AM) to measure the pH of the mitochondrial matrix. Subsequent flow cytometry studies were designed to evaluate the effect in mitochondria function of two well-known apoptosis inducers in both mammalian cell lines and yeast cells, namely bovine lactoferrin (bLf) and acetate, and to understand whether or not they act directly on mitochondria independently of upstream cellular pathways. The results showed that both bLf and acetate promoted changes in mitochondrial mass, matrix mitochondrial pH, Δψm and ROS levels of rat liver mitochondria. Moreover, the assessment of the activity of the complexes of electron transport chain (ETC) showed that bLf and acetate affect their activity and trigger mitochondrial swelling. These results are particularly interesting since the two compounds appear to induce in isolated mitochondria mainly the same alterations as those occurring in whole cells, and which have been proposed to be involved in the induction of cell death. Altogether, this study show that flow cytometry is a valuable tool to study key mitochondrial functional parameters and to screen for mitochondriatargeted drugs, including the anticancer agents, such as acetate and bLf. |
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