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Difference between revisions of "Bufe 2017 TRACT PhD project abstract"

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Metabolic transformation is a universal property of tumor formation and is a rich source of targets for development of therapeutic interventions 1. Pilot studies performed in the Queen’s University of Belfast (QUB) using a pathway based approach to identify determinants of drug resistance in oral and oesophageal cancer (OOC) have identified the glycolytic pathway as a potential targetable pathway. Hence, this work will further characterize the bioenergetic and metabolic properties of normal, dysplastic and cancerous oral cancer cells and identify differences between the different stages of carcinogenesis. The study will include comparison of oxygen consumption, extracellular acidity and metabolic flux under normoxic and hypoxic conditions. The measurements will be conducted using the Oroboros Respirometer Multisensor system Oxygraph2k (O2k) enabling the screening of real-time bioenergetics and metabolism in the different stages of OCC. Due to the combination of high resolution respirometry (HRR) with optical methods, namely fluorometry and spectrophotometry, in the oxygraph, besides cellular respiration, cellular functions and metabolic parameters including mitochondrial membrane potential, ATP production (indirectly via the concentration of free Mg<sup>2+</sup>), Ca<sup>2+</sup> production and reactive oxygen (ROS) production (via H<sub>2</sub>O<sub>2</sub> concentration) will be analyzed. These parameters are often altered in cancerous cells and might therefore be interesting for diagnostic and therapeutic interventions. By inhibiting or upregulating the activity of specific enzymes or complexes of the respiratory chain, their functions in the progression of cancer might be proved and compared to other cell types. To name an example, the production of ROS is known to be an essential component of multiple cellular pathways; however, an overload of oxidative stress can also cause genetic and functional damage, leading to degenerative diseases or cancer. By measuring the oxidative stress as a signal of hydrogen peroxide production and decreasing/increasing it by the addition of specific chemicals, it could be further evaluated how significant the effects of ROS production might be in the development of OOC.
Metabolic transformation is a universal property of tumor formation and is a rich source of targets for development of therapeutic interventions 1. Pilot studies performed in the Queen’s University of Belfast (QUB) using a pathway based approach to identify determinants of drug resistance in oral and oesophageal cancer (OOC) have identified the glycolytic pathway as a potential targetable pathway. Hence, this work will further characterize the bioenergetic and metabolic properties of normal, dysplastic and cancerous oral cancer cells and identify differences between the different stages of carcinogenesis. The study will include comparison of oxygen consumption, extracellular acidity and metabolic flux under normoxic and hypoxic conditions. The measurements will be conducted using the Oroboros Respirometer Multisensor system Oxygraph2k (O2k) enabling the screening of real-time bioenergetics and metabolism in the different stages of OCC. Due to the combination of high resolution respirometry (HRR) with optical methods, namely fluorometry and spectrophotometry, in the oxygraph, besides cellular respiration, cellular functions and metabolic parameters including mitochondrial membrane potential, ATP production (indirectly via the concentration of free Mg<sup>2+</sup>), Ca<sup>2+</sup> production and reactive oxygen (ROS) production (via H<sub>2</sub>O<sub>2</sub> concentration) will be analyzed. These parameters are often altered in cancerous cells and might therefore be interesting for diagnostic and therapeutic interventions. By inhibiting or upregulating the activity of specific enzymes or complexes of the respiratory chain, their functions in the progression of cancer might be proved and compared to other cell types. To name an example, the production of ROS is known to be an essential component of multiple cellular pathways; however, an overload of oxidative stress can also cause genetic and functional damage, leading to degenerative diseases or cancer. By measuring the oxidative stress as a signal of hydrogen peroxide production and decreasing/increasing it by the addition of specific chemicals, it could be further evaluated how significant the effects of ROS production might be in the development of OOC.
Over the course of a 9-month secondment at the Trinity College of Dublin (TCD) under the supervision of Prof. Richard Porter, metabolic flux will be measured additionally through glycolysis, pentose phosphate pathway and glutaminolysis using 2H/13C NMR to complement the respiration/fluorometric measurements conducted with the O2k at OROBOROS INSTRUMENTS in Innsbruck.
Over the course of a 9-month secondment at the Trinity College of Dublin (TCD) under the supervision of Prof. Richard Porter, metabolic flux will be measured additionally through glycolysis, pentose phosphate pathway and glutaminolysis using <sup>2</sup>H/<sup>13</sup>C NMR to complement the respiration/fluorometric measurements conducted with the O2k at OROBOROS INSTRUMENTS in Innsbruck.


The stated experiments and investigations will be performed with cell culture models of oesophageal adenocarcinoma, provided by TCD, as well as human tissue biopsies of different stages of cancer (intestinal metaplasia, dysplasia and adenocarcinoma) and non-cancerous tissue, which will be provided by the surgery of Innsbruck (UniversitĂ€tsklinik fĂŒr Visceral-, Transplantations- und Thoraxchirurgie der Medizinische UniversitĂ€t Innsbruck). For the conduction of the experiments, an ethics committee vote is obtained. To enable the repeated measurement of the samples, parts of all received samples are intended to be cryopreserved. Anyway, the feasibility of the cryopreservation of these tissues remains to be tested and will be proven by simultaneous comparative measurements of the cellular functions and metabolism of cryopreserved as well as fresh tissues with the O2k.
The stated experiments and investigations will be performed with cell culture models of oesophageal adenocarcinoma, provided by TCD, as well as human tissue biopsies of different stages of cancer (intestinal metaplasia, dysplasia and adenocarcinoma) and non-cancerous tissue, which will be provided by the surgery of Innsbruck (UniversitĂ€tsklinik fĂŒr Visceral-, Transplantations- und Thoraxchirurgie der Medizinische UniversitĂ€t Innsbruck). For the conduction of the experiments, an ethics committee vote is obtained. To enable the repeated measurement of the samples, parts of all received samples are intended to be cryopreserved. Anyway, the feasibility of the cryopreservation of these tissues remains to be tested and will be proven by simultaneous comparative measurements of the cellular functions and metabolism of cryopreserved as well as fresh tissues with the O2k.

Revision as of 10:26, 28 March 2017

Metabolic profiles in normal, dysplastic and cancerous oral cells.

Link:

Bufe A (2017)

Event: TRACT

The stated project will be conducted in the frame of the European Union's TRACT program, funded by a Marie SkƂodowska-Curie fellowship. With the company OROBOROS INSTRUMENTS as a partner, it will aim at the examination of metabolic transformation mechanisms in oesophageal cancer (OOC) with the attempt of identifying new drug targets for future therapeutic development.

Metabolic transformation is a universal property of tumor formation and is a rich source of targets for development of therapeutic interventions 1. Pilot studies performed in the Queen’s University of Belfast (QUB) using a pathway based approach to identify determinants of drug resistance in oral and oesophageal cancer (OOC) have identified the glycolytic pathway as a potential targetable pathway. Hence, this work will further characterize the bioenergetic and metabolic properties of normal, dysplastic and cancerous oral cancer cells and identify differences between the different stages of carcinogenesis. The study will include comparison of oxygen consumption, extracellular acidity and metabolic flux under normoxic and hypoxic conditions. The measurements will be conducted using the Oroboros Respirometer Multisensor system Oxygraph2k (O2k) enabling the screening of real-time bioenergetics and metabolism in the different stages of OCC. Due to the combination of high resolution respirometry (HRR) with optical methods, namely fluorometry and spectrophotometry, in the oxygraph, besides cellular respiration, cellular functions and metabolic parameters including mitochondrial membrane potential, ATP production (indirectly via the concentration of free Mg2+), Ca2+ production and reactive oxygen (ROS) production (via H2O2 concentration) will be analyzed. These parameters are often altered in cancerous cells and might therefore be interesting for diagnostic and therapeutic interventions. By inhibiting or upregulating the activity of specific enzymes or complexes of the respiratory chain, their functions in the progression of cancer might be proved and compared to other cell types. To name an example, the production of ROS is known to be an essential component of multiple cellular pathways; however, an overload of oxidative stress can also cause genetic and functional damage, leading to degenerative diseases or cancer. By measuring the oxidative stress as a signal of hydrogen peroxide production and decreasing/increasing it by the addition of specific chemicals, it could be further evaluated how significant the effects of ROS production might be in the development of OOC. Over the course of a 9-month secondment at the Trinity College of Dublin (TCD) under the supervision of Prof. Richard Porter, metabolic flux will be measured additionally through glycolysis, pentose phosphate pathway and glutaminolysis using 2H/13C NMR to complement the respiration/fluorometric measurements conducted with the O2k at OROBOROS INSTRUMENTS in Innsbruck.

The stated experiments and investigations will be performed with cell culture models of oesophageal adenocarcinoma, provided by TCD, as well as human tissue biopsies of different stages of cancer (intestinal metaplasia, dysplasia and adenocarcinoma) and non-cancerous tissue, which will be provided by the surgery of Innsbruck (UniversitĂ€tsklinik fĂŒr Visceral-, Transplantations- und Thoraxchirurgie der Medizinische UniversitĂ€t Innsbruck). For the conduction of the experiments, an ethics committee vote is obtained. To enable the repeated measurement of the samples, parts of all received samples are intended to be cryopreserved. Anyway, the feasibility of the cryopreservation of these tissues remains to be tested and will be proven by simultaneous comparative measurements of the cellular functions and metabolism of cryopreserved as well as fresh tissues with the O2k.

By comparing the different types of cancerous tissue with the cell culture model of adenocarcinoma, the representativeness of the utilized cell model can be confirmed and adopted or modified if necessary. By including human tissue in this study, the relevance and applicability for the clinic is ensured.

By identifying the differences in the metabolic profiles of the stated cells and metabolic transformation mechanisms in OOC, differential novel drug targets and means to enhance the chemotherapeutic sensitivity of cancer cells are intended to be identified.


‱ O2k-Network Lab: AT Innsbruck OROBOROS


Labels: MiParea: Respiration  Pathology: Cancer 

Organism: Human  Tissue;cell: Other cell lines  Preparation: Intact cells, Permeabilized cells, Permeabilized tissue 



HRR: Oxygraph-2k, O2k-Fluorometer